Preparation method of enzyme mutant and snake venom-like tripeptide

Abstract

The present invention relates to the field of biotechnology, and in particular to methods for preparing enzyme mutants and snake venom-like tripeptides. The present invention provides amino acid ligase mutants, including PDLigA or APDLigB, and a fusion protein of the two, APDLigAB. Also provided are uses of the mutants or fusion proteins in preparing snake venom-like tripeptides, and methods for preparing the snake venom-like tripeptides. This method exhibits significant advantages in terms of production cost, energy consumption, product quality, and green performance.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a preparation method of an enzyme mutant and a snake venom-like tripeptide. Background Art

[0002] Tripeptide-3 is a highly effective wrinkle-reducing peptide commonly used in cosmetics. Its sequence is β-Ala-Pro-Dab-NHBn, also known as dipeptide diaminobutyric acid benzyl salt or SYN-AKE. Tripeptide-3 is a small peptide that mimics the activity of Waglerin I, a toxin found in the venom of the highly toxic Tropidolaemus wagleri. It acts on the postsynaptic membrane and is a reversible antagonist of acetylcholine receptors. Research results have shown that using a 4% solution of tripeptide-3 for 28 days can reduce facial wrinkles by 52%. Therefore, tripeptide-3 is a timely or long-term solution for the elimination of fine and coarse wrinkles. Tripeptide-3 also protects against oxidative stress and inflammatory damage to vascular endothelial cells.

[0003] Preparation method of snake venom peptide:

[0004] Currently, the preparation methods of snake venom-like tripeptides are all chemical synthesis methods, and the chemical methods are divided into solid phase synthesis and liquid phase synthesis methods.

[0005] This type of method requires protected amino acids as raw materials, followed by individual condensation reactions, and finally, the C-terminus is modified with benzylamine. Continuous coupling and deprotection steps are required during the specific process, resulting in cumbersome steps, low overall yield, and low product purity. Of particular note is the tendency for Dab racemization to occur during the chemical condensation process, a process that significantly impacts the quality of the final product. The chemical coupling and deprotection processes inevitably involve the use of large amounts of organic solvents and even heavy metals, posing potential safety risks to the environment and the product.

[0006] As mentioned above, the current industrial production method for snake venom-like tripeptides is primarily chemical synthesis. However, because chemical synthesis requires the use of protected amino acids and chemical coupling, the overall preparation route is long and the yield is low. Furthermore, the production process requires the use of organic solvents and heavy metals, resulting in significant environmental pollution. The racemization of amino acids caused by coupling during the chemical preparation process also significantly affects the quality of the snake venom-like tripeptide products. Summary of the Invention

[0007] In light of this, the present invention provides methods for preparing enzyme mutants and snake venom-like tripeptides. These methods pioneer the use of biological enzymes as catalysts to link three raw amino acids in aqueous solution with high yields. The reaction conditions are mild, the operation is simple, and no impurities are generated. The snake venom-like tripeptide preparation method demonstrated in this invention offers significant advantages.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a mutant of amino acid ligase, comprising:

[0010] (I) Mutant 1, comprising a mutation of aspartic acid at position 13 of the amino acid ligase with Uniprot ID A0A1H0FZL2 to histidine, glutamine at position 81 to aspartic acid, proline at position 83 to cysteine, valine at position 85 to leucine, isoleucine at position 238 to alanine, aspartic acid at position 240 to glutamic acid, proline at position 241 to threonine, threonine at position 288 to phenylalanine, glycine at position 290 to glutamine, valine at position 292 to isoleucine, and valine at position 336 to glycine;

[0011] or

[0012] (II), mutant 2, comprising mutations of proline at position 10 to glycine, serine at position 78 to histidine, tyrosine at position 80 to isoleucine, leucine at position 82 to alanine, valine at position 237 to methionine, aspartic acid at position 240 to threonine, valine at position 241 to isoleucine, glycine at position 284 to threonine, alanine at position 286 to glutamic acid, valine at position 288 to tyrosine, and glycine at position 332 to asparagine of the amino acid ligase with Uniprot ID A0A2N7GIY4;

[0013] or

[0014] (III), a fusion protein of the mutant 1 and the mutant 2;

[0015] The fusion protein comprises one or more connecting peptides connecting the mutant 1 and the mutant 2;

[0016] The several are 2 to 10.

[0017] In some specific embodiments of the present invention, the mutants include:

[0018] The mutant 1 has:

[0019] (1) the amino acid sequence shown in SEQ ID No. 4; or

[0020] (2) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or

[0021] (3) an amino acid sequence having at least 70% homology to the amino acid sequence shown in (1) or (2);

[0022] or

[0023] The mutant 2 has:

[0024] (4) the amino acid sequence shown in SEQ ID No. 7; or

[0025] (5) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (4), and having the same or similar function as (4); or

[0026] (6) an amino acid sequence having at least 70% homology to the amino acid sequence shown in (4) or (5);

[0027] or

[0028] The fusion protein has:

[0029] (7), the amino acid sequence shown in SEQ ID No. 8; or

[0030] (8) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (7), and having the same or similar function as (7); or

[0031] (9) an amino acid sequence having at least 70% homology to the amino acid sequence shown in (7) or (8);

[0032] The plurality is 2 to 250;

[0033] or

[0034] The connecting peptide has:

[0035] (10), the amino acid sequence shown in SEQ ID No. 13; or

[0036] (11) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (10), and having the same or similar function as (10); or

[0037] (12) an amino acid sequence having at least 70% homology to the amino acid sequence shown in (10) or (11);

[0038] The several are 2 to 10.

[0039] The present invention also provides a nucleic acid molecule encoding the above mutant;

[0040] or

[0041] The nucleic acid molecule has:

[0042] (13) a nucleotide sequence as shown in any one of SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 12; or

[0043] (14) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (13), and having the same or similar function as (13); or

[0044] (15) A nucleotide sequence having at least 70% homology to the nucleotide sequence shown in (13) or (14);

[0045] The plurality is from 2 to 700.

[0046] The present invention also provides an expression vector comprising the above nucleic acid molecule and acceptable gene elements.

[0047] The present invention also provides a host cell comprising the above nucleic acid molecule or the above expression vector.

[0048] The present invention also provides a composition comprising the mutant and adenosine triphosphate.

[0049] In some specific embodiments of the present invention, the above composition further comprises polyphosphate kinase or polyphosphate;

[0050] The polyphosphoric acid includes hexaphosphate.

[0051] In some specific embodiments of the present invention, the polyphosphate kinase in the above composition has:

[0052] (16), the amino acid sequence shown in SEQ ID No. 1; or

[0053] (17) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (16), and having the same or similar function as (16); or

[0054] (18) an amino acid sequence having at least 70% homology to the amino acid sequence shown in (16) or (17);

[0055] The plurality is 2 to 80.

[0056] The present invention also provides the use of the mutant 1 among the above mutants in the preparation of Pro-Dab-Bn.

[0057] The present invention also provides the use of any one of the following in synthesizing snake venom-like tripeptides:

[0058] (i) the above mutants;

[0059] (ii) the nucleic acid molecule described above;

[0060] (iii) the above-mentioned expression vector;

[0061] (iv) the above-mentioned host cell;

[0062] (v) The above composition.

[0063] The present invention also provides a method for preparing the snake venom-like tripeptide, comprising:

[0064] (a) mixing the amino acid with the mutant 1 and the mutant 2 among the above mutants to obtain a snake venom-like tripeptide; or

[0065] (b) mixing amino acids, Pro-Dab-Bn and the mutant 2 among the above mutants to obtain a snake venom-like tripeptide; or

[0066] (c) mixing the amino acids and the fusion protein in the mutant to obtain a snake venom-like tripeptide; or

[0067] (d) expressing the nucleic acid molecule, and mixing the obtained protein product with amino acids to obtain a snake venom-like tripeptide; or

[0068] (e) expressing the above expression vector, and mixing the obtained protein product with amino acids to obtain a snake venom-like tripeptide; or

[0069] (f) culturing the host cell, mixing the obtained protein product with amino acids to obtain a snake venom-like tripeptide; or

[0070] (g) mixing the amino acid and the above composition to obtain a snake venom-like tripeptide;

[0071] The amino acids include β-ala, L-Pro and / or Dab-Bn.

[0072] The enzyme mutant and the preparation method of the snake venom-like tripeptide of the present invention have the following effects:

[0073] The present invention discovered that the amino acid ligase from Actinobacterium persimmon has the ability to connect L-proline and 1,4-diaminobutyric acid benzyl ester to form the Pro-Dab-Bn dipeptide, and that the amino acid ligase from Vibrio lentiviralus can combine Pro-Dab-Bn with β-alanine to form a snake venom-like tripeptide. By modifying these two enzymes, their catalytic activity is significantly improved, enabling the ligation of three amino acids into a snake venom-like tripeptide at high concentration and high yield. Compared to commercially available chemical synthesis processes, this method offers significant advantages in production cost, energy consumption, product quality, and environmental performance. Therefore, this method is the optimal choice for large-scale production of these snake venom-like tripeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0075] Figure 1 The Pro-Dab-Bn NMR spectrum is shown; its data are: 1 H NMR(400MHz,D2O)δ7.55–7.06(m,5H),4.50–4.32(m,4H),3.61(t,J=6.7Hz,2H),3.1 7–3.05(m,2H),2.33-2.13(m,3H),2.01-1.85(m,3H).;MS(ESI):m / z:305.2[M+H]+;

[0076] Figure 2 The formation of the liquid phase detection product after the reaction of Example 2 is shown;

[0077] Figure 3 HPLC purity test of the product of Example 2 is shown;

[0078] Figure 4 2. Mass spectrometry detection of the product of Example 2 is shown;

[0079] Figure 5 The β-Ala-Pro-Dab-Bn NMR spectrum is shown; the data are: 1 H NMR(400MHz,D2O)δ7.52–7.08(m,5H),4.52–4.36(m,4H),3.63(t,J=6.7Hz,2H),3.21(t,J=6 .5Hz,2H),3.16–3.05(m,2H),2.85(t,J=6.5Hz,2H),2.31-2.13(m,3H),2.02-1.83(m,3H).;

[0080] Figure 6 The gel image of the protein used in the patent is shown; the left lane is the marker. DETAILED DESCRIPTION

[0081] The present invention discloses methods for preparing enzyme mutants and snake venom-like tripeptides. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications obvious to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art can modify or appropriately alter and combine the methods and applications described herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0082] Literature reports indicate that an amino acid ligase (Uniprot: Q842E2) from Pseudomonas syringae can link a wide variety of amino acids and, through simple mutations, can synthesize L-proline-containing dipeptides. Following this lead, through NCBI gene library analysis and experimental verification, we identified a Q842E2 homolog, A0A1H0FZL2, from Actinokineospora alba, which exhibits weak activity in catalyzing the ligation of Pro-Dab-Bn. Furthermore, we discovered a ligase (Uniprot: A0A2N7GIY4) from Vibrio lentus that can conjugate proline-containing dipeptides to β-alanine. Building on this, we generated highly active ligases (PDLigA & APDLigB) by mutagenesis of these two enzyme systems. These ligases can convert three amino acids (β-ala, L-pro, and Dab-Bn) into snake venom-like tripeptides with high yields.

[0083] The patented amino acid ligase method for preparing snake venom-like tripeptides is as follows:

[0084]

[0085] This route utilizes inexpensive, unprotected amino acids as starting materials, which are converted into the corresponding snake venom-like tripeptides in high yields using an equivalent amount of adenosine triphosphate (ATP) and the corresponding amino acid ligase. To further reduce production costs, the amount of ATP in the reaction system can be reduced by utilizing a recycling system. Furthermore, the amount of enzyme required for specific production can be further reduced by constructing fusion enzymes or immobilized enzymes. Therefore, compared to the chemical synthesis process for snake venom-like tripeptides, this patented preparation route is streamlined, efficient, and produces high-quality products. It also features a high green index during production and is easily scalable.

[0086] The present invention utilizes a corresponding ligase to directly ligate two or three target amino acid raw materials into the corresponding dipeptide / tripeptide products in a single step in a buffer. The required adenosine triphosphate (ATP) can be an equivalent amount or a catalytic amount (by using an ATP regeneration system, such as polyphosphate kinase (PPK) and hexaphosphate), and the enzyme used can be a crude enzyme solution or an immobilized enzyme.

[0087] The SDS-PAGE results of the enzymes used in the present invention are shown in Figure 6 .

[0088] The enzyme-related information of the present invention is as follows:

[0089] Ligase PDLigA0: The parent enzyme is derived from Actinokineospora alba (UniprotID: A0A1H0FZL2) and has no mutation.

[0090] Ligase PDLigA5: The fifth mutant. The catalytic performance did not meet expectations. The mutations are: D13H, P83C, V85L, I238A, P241T, T288F, G290Q, V292I.

[0091] Ligase PDLigA: The parent is derived from Actinokineospora alba (UniprotID: A0A1H0FZL2), and the mutation sites are: D13H, Q81D, P83C, V85L, I238A, D240E, P241T, T288F, G290Q, V292I, V336G.

[0092] Ligase APDLigB0: The parent protein is derived from Vibrio lentus (Uniprot ID: A0A2N7GIY4); no mutations.

[0093] Ligase APDLigB13: Mutant #13. Catalytic activity did not meet expectations. Mutations are: P10G, S78H, Y80I, V237M, G284T, A286E.

[0094] Ligase APDLigB: The parent strain is derived from Vibrio lentus (Uniprot ID: A0A2N7GIY4); the mutation sites are: P10G, S78H, Y80I, L82A, V237M, D240T, V241I, G284T, A286E, V288Y, G332N.

[0095] Ligase APDLigAB: PDLigA and APDLigB are connected through gene-level construction, and the connecting peptide sequence is: GGGGS EAAAK EAAAK GGGGS (SEQ ID No. 13).

[0096] Polyphosphate kinase (PPK): derived from Rhodopseudomonas palustris (Uniprot ID: Q6N140)

[0097] The sequence information involved in the present invention is:

[0098] Table 1

[0099]

[0100]

[0101] Table 2

[0102]

[0103]

[0104]

[0105] The present invention relates to a fermentation production method of an enzyme:

[0106] The enzymes required for this patent are synthesized by a service company, then constructed onto a specific expression plasmid and produced through E. coli fermentation. The process specifically includes the following steps: The genes corresponding to the above enzymes are sequence-optimized and then ordered to General Biotechnology (Chuzhou, Anhui) for synthesis. NdeI / XhoI restriction sites are then introduced and subcloned into the pET28a expression vector. Plasmids with confirmed sequence accuracy are then transferred into E. coli (BL21) competent cells for plate culture (Qingke Biotechnology) and small-scale liquid culture of single clones. Bacteria with correct protein expression are then scaled up for liquid culture. Specifically, a single colony is transferred into 5 mL of LB culture medium containing 50 μM kanamycin (37°C) for culture. When the cells reach the logarithmic phase, they are inoculated into 250 mL of LB culture medium containing the same antibiotic. When they also reach the logarithmic phase, they are transferred to a 5 L fermenter for culture and final protein expression. In a 5L fermentor, when the cell OD value reached ~20, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added at 25°C to induce protein expression for 6 hours. Finally, the cells were harvested by high-speed centrifugation (4000 rpm, 20 minutes) to obtain 25-50 g of enzyme-overexpressing wet cells. A small amount of cells was first mixed with Tris-HCl buffer (50 mM, pH 8.0) on ice. The cells were then disrupted by freeze-thaw, centrifuged at high speed to remove the cell wall, and the supernatant was run on SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to determine protein expression. Bacterial cells expressing the correct protein were used for the next catalytic experiment. Specifically, the remaining cells were mixed with Tris-HCl buffer (50 mM, pH 8.0) at low temperature (~10 g of wet cells: 200 mL of buffer). The cell walls were then disrupted by low-temperature high pressure. After high-speed centrifugation (16,000 rpm, 45 min), the enzyme-containing supernatant was obtained for use (the resulting enzyme activity was 400-700 U / mL, where U is the amount of enzyme required to convert 1 μmol of substrate per minute at room temperature). LB medium was composed of: 1% tryptone, 0.5% yeast extract, 1% NaCl, 1% dipotassium phosphate, 1% dipotassium phosphate, and 5% glycerol.

[0107] The present invention relates to an enzyme immobilization method:

[0108] Solid ammonium sulfate was slowly added to the crude enzyme supernatant collected above until protein solids precipitated (20-50% w / v ammonium sulfate:buffer). The protein solids were collected by high-speed centrifugation (10,000 rpm, 10 minutes) and slowly dissolved in 25 mM Tris buffer (pH 8.0) (buffer A). The enzyme solution was then dialyzed against 50 volumes of buffer A (twice, with a 4-hour interval between each) to remove ammonium sulfate. Finally, the dialyzate was loaded onto a DEAE Seplite FF (Xi'an Lanxiao Company) anion exchange column (NaCl gradient elution in buffer: 0-1N NaCl) to obtain pre-purified APDLigAB and PPK enzyme solutions. The APDLigAB and PPK enzymes were then immobilized using LX-1000EP epoxy resin (Xi'an Lanxiao Company) at an activity unit ratio of 1:(2-3) using the following method: 2000 U of the purified enzyme mixture was dissolved in 1 L of 50 mM pH 8.0. 8.0 potassium phosphate solution (buffer B), followed by the addition of 30-50 mM phenoxyacetic acid and 500 g of LX-1000EP epoxy resin. After stirring at room temperature for 10 hours, the immobilized enzyme was filtered out and finally washed twice with water and buffer B, respectively, and stored at low temperature until use. The immobilized enzyme had an initial activity of 70-92%.

[0109] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0110] The present invention will be further described below in conjunction with the embodiments:

[0111] Example 1: Preparation of Pro-Dab-Bn using ligase PDLigA

[0112]

[0113] To 1 L of 100 mM Tris-HCl (pH 8.0) solution, 20.7 g of 1,4-diaminobenzyl ester Dab-Bn (100 mM), 12.1 g of L-proline (105 mM), and 58.2 g of adenosine triphosphate monosodium salt (ATP, 110 mM) were added. The pH of the reaction system was then adjusted to 8.0 with aqueous NaOH solution. The ligase PDLigA1500U was then added to initiate the reaction. The reaction was gently stirred at room temperature (25°C) while maintaining the pH of the reaction system between 7.0 and 9.0. After 3 hours, HPLC analysis indicated that the reaction of the raw materials was essentially complete. The pH of the reaction solution was then adjusted to 1.0 with aqueous HCl to denature the enzyme in the reaction system and remove the precipitate by centrifugation. The pH of the reaction solution was then adjusted to 7.0 and directly loaded onto D201 anion exchange resin to remove adenosine diphosphate and free phosphate impurities. The crude product was finally desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol: water, 2:1, v:v) to obtain 24.9 g of Pro-Dab-Bn dipeptide (yield 82%). The structure was confirmed by NMR analysis. Figure 1 The data is: 1 H NMR(400MHz,D2O)δ7.55–7.06(m,5H),4.50–4.32(m,4H),3.61(t,J=6.7Hz,2H),3.17–3.05(m,2H),2.33-2.13(m,3H),2.01-1.85(m,3H).

[0114] Example 2: Preparation of snake venom-like tripeptides using ligase APDLigB

[0115]

[0116] Similar to the above-mentioned preparation method of Pro-Dab-Bn, 30.4 g of Pro-Dab-Bn dipeptide (100 mM), 9.8 g of β-alanine (110 mM), and 58.2 g of adenosine triphosphate monosodium salt (ATP, 110 mM) were added to 1 L of 100 mM Tris-HCl solution (pH 8.0). The pH of the reaction system was then adjusted to 8.0 with aqueous NaOH solution. 2000 U of ligase APDLigB was added to initiate the reaction. The reaction was stirred gently at 30° C. and the pH of the reaction system was maintained between 7.0 and 9.0. After 2 hours, the reaction of the raw materials was basically complete as detected by HPLC, as shown in the liquid phase analysis. Figure 2The pH of the reaction solution was then adjusted to 1.0 with HCl aqueous solution to denature the enzyme in the reaction system and remove the precipitate by centrifugation. The pH of the reaction solution was then adjusted to 7.0 and directly loaded onto D201 anion exchange resin to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol: water, 1:1, v:v) to obtain 32.6 g of snake venom tripeptide (β-Ala-Pro-Dab-Bn, yield 87%) with an HPLC purity of 100% ( Figure 3 ), mass spectrometry confirmed MS: 376.2 ( Figure 4 ). And send samples for NMR to confirm the structure ( Figure 5 ): 1 H NMR(400MHz,D2O)δ7.52–7.08(m,5H),4.52–4.36(m,4H),3.63(t,J=6.7Hz,2H),3.21(t,J= 6.5Hz,2H),3.16–3.05(m,2H),2.85(t,J=6.5Hz,2H),2.31-2.13(m,3H),2.02-1.83(m,3H).

[0117] Example 3: One-step generation of snake venom-like tripeptides using ligase APDLigAB

[0118]

[0119] Similarly, 20.7 g of 1,4-diaminobenzyl ester Dab-Bn (100 mM), 12.1 g of L-proline (105 mM), 9.8 g of β-alanine (110 mM), and 116.4 g of adenosine triphosphate monosodium salt (ATP, 220 mM) were added to 1 L of 100 mM Tris-HCl solution (pH 8.0). The pH of the reaction system was then adjusted to 8.0 with aqueous NaOH solution, and ligase APDLigAB 3000 U was added to initiate the reaction. The reaction was gently stirred at 30° C. and the pH of the reaction system was maintained between 7.0 and 9.0. After 4 hours, the reaction of the raw materials was basically complete by HPLC. The pH was then adjusted to 1.0 with aqueous HCl to denature the enzyme in the reaction system and remove the precipitate by centrifugation. The pH of the reaction solution was then adjusted to 7.0 and directly loaded onto a D201 anion exchange resin to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 1:1, v:v) to obtain 27.3 g of a snake venom-like tripeptide (yield 73%).

[0120] Example 4: One-step generation of snake venom-like tripeptides using ligase APDLigAB and ATP regeneration system

[0121]

[0122] Similarly, to 1 L of 100 mM Tris-HCl (pH 7.5), add 20.7 g of 1,4-diaminobenzyl ester Dab-Bn (100 mM), 12.1 g of L-proline (105 mM), 9.8 g of β-alanine (110 mM), 5.4 g of adenosine triphosphate monosodium salt (ATP, 10 mM), and 32.2 g of sodium metabisphosphate (52.6 mM). Adjust the pH of the solution to 7.5, then add 2000 U of APDLigAB and 3000 U of polyphosphate kinase (PPK) to initiate the reaction. Gently stir at 30°C while maintaining the pH between 7.0 and 9.0. After 3 hours, HPLC analysis indicates that the starting materials are essentially reacted. The pH was then adjusted to 1.0 with aqueous HCl to denature the enzyme in the reaction system and remove the precipitate by centrifugation. The pH of the reaction solution was then adjusted to 7.0 and directly loaded onto a D201 anion exchange resin to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 1:1, v:v) to obtain 29.6 g of a snake venom-like peptide (yield 79%).

[0123] Example 5: One-step generation of snake venom-like tripeptides using immobilized APDLigAB and PPK

[0124]

[0125] The reaction was similar to that in Example 4 above. To 1 L of a 100 mM Tris-HCl solution (pH 8.0) containing 100 mM 1,4-diaminobenzyl ester (Dab-Bn) (100 mM), 12.1 g L-proline (105 mM), 9.8 g β-alanine (110 mM), 5.4 g adenosine triphosphate monosodium salt (ATP, 10 mM), and 32.2 g sodium metabisphosphate (52.6 mM) was added. The pH of the solution was adjusted to 8.0, and then the mixed immobilized APDLigAB / PPK (4000 U total activity) was added. The reaction was stirred gently at 30°C, maintaining the pH between 7.0 and 9.0. After 4 hours, HPLC analysis confirmed that the reaction was substantially complete. The immobilized APDLigAB / PPK enzyme was then directly recovered by filtration, and the solid was rinsed three times with 25 mM Tris pH 8.0 buffer for later use. The reaction solution was adjusted to pH 1.0 with aqueous HCl to precipitate protein, which was then removed by centrifugation. The pH of the reaction solution was then adjusted to pH 7.0 and directly loaded onto a D201 anion exchange resin to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 1:1, v:v) to obtain 32.6 g of a snake venom tripeptide (yield 87%). The recovered immobilized APDLigAB / PPK enzyme had 91% of its initial activity.

[0126] Comparative Example 1: Preparation of Pro-Dab-Bn using ligase PDLigA0

[0127]

[0128] To 1 L of 100 mM Tris-HCl (pH 8.0) solution, 20.7 g of 1,4-diaminobenzyl ester Dab-Bn (100 mM), 12.1 g of L-proline (105 mM), and 58.2 g of adenosine triphosphate monosodium salt (ATP, 110 mM) were added. The pH of the reaction system was then adjusted to 8.0 with aqueous NaOH solution. The reaction was initiated by adding 1500 U of ligase PDLigA0. The reaction was gently stirred at room temperature (25°C) while maintaining the pH of the reaction system between 7.0 and 9.0. After 3 hours, HPLC analysis revealed that only 20% of the raw material had been converted.

[0129] Comparative Example 2: Preparation of Pro-Dab-Bn using ligase PDLigA5

[0130]

[0131] To 1 L of 100 mM Tris-HCl (pH 8.0) solution, 20.7 g of 1,4-diaminobenzyl ester Dab-Bn (100 mM), 12.1 g of L-proline (105 mM), and 58.2 g of adenosine triphosphate monosodium salt (ATP, 110 mM) were added. The pH of the reaction system was then adjusted to 8.0 with aqueous NaOH solution. 1500 U of ligase PDLigA0 was added to initiate the reaction. The reaction was gently stirred at room temperature (25°C) while maintaining the pH of the reaction system between 7.0 and 9.0. After 3 hours, HPLC analysis revealed that approximately 50% of the raw material had been converted.

[0132] Comparative Example 3: Preparation of snake venom-like tripeptides using ligase APDLigB0

[0133]

[0134] Similar to the preparation method in Comparative Example 1 above, 30.4 g of Pro-Dab-Bn dipeptide (100 mM), 9.8 g of β-alanine (110 mM), and 58.2 g of adenosine triphosphate monosodium salt (ATP, 110 mM) were added to 1 L of 100 mM Tris-HCl solution (pH 8.0). The pH of the reaction system was then adjusted to 8.0 with an aqueous NaOH solution. Ligase APDLigB 2000 U was added to initiate the reaction. The reaction was gently stirred at 30° C. and the pH of the reaction system was maintained between 7.0 and 9.0. After 2 hours, HPLC detected that approximately 35% of the raw material was converted into the product.

[0135] Comparative Example 4: Preparation of snake venom-like tripeptides using ligase APDLigB13

[0136]

[0137] Similar to the preparation method in Comparative Example 3 above, 30.4 g of Pro-Dab-Bn dipeptide (100 mM), 9.8 g of β-alanine (110 mM), and 58.2 g of adenosine triphosphate monosodium salt (ATP, 110 mM) were added to 1 L of 100 mM Tris-HCl solution (pH 8.0). The pH of the reaction system was then adjusted to 8.0 with an aqueous NaOH solution. 2000 U of ligase APDLigB was added to initiate the reaction. The reaction was stirred gently at 30° C. and the pH of the reaction system was maintained between 7.0 and 9.0. After 2 hours, approximately 68% of the raw material was converted into the product as detected by HPLC.

[0138] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A mutant of an amino acid ligase, characterized in that The mutants are: (I), mutant 1, the amino acid sequence of which is shown in SEQ ID No. 4; or (II), mutant 2, the amino acid sequence of which is shown in SEQ ID No. 7; or (III), a fusion protein of the mutant 1 and the mutant 2; The amino acid sequence of the fusion protein is shown in SEQ ID No.

8.

2. A nucleic acid molecule, characterized in that Encoding the mutant according to claim 1; The nucleotide sequence of the nucleic acid molecule is shown in any one of SEQ ID No.10, SEQ ID No.11 or SEQ ID No.

12.

3. An expression vector, characterized in that The method comprises the nucleic acid molecule according to claim 2, and an acceptable genetic element.

4. A host cell, characterized in that comprising the nucleic acid molecule according to claim 2 or the expression vector according to claim 3.

5. A composition, characterized in that The method comprises the mutant according to claim 1 and adenosine triphosphate.

6. The composition according to claim 5, wherein Also included are polyphosphate kinase and polyphosphate; The polyphosphoric acid includes hexaphosphate.

7. The composition according to claim 6, wherein The amino acid sequence of the polyphosphate kinase is shown in SEQ ID No.

1.

8. Use of the mutant 1 among the mutants according to claim 1 in the preparation of Pro-Dab-Bn.

9. Use of any of the following in the synthesis of β-Ala-Pro-Dab-Bn: (i) The mutant according to claim 1; (ii) the nucleic acid molecule according to claim 2; (iii) the expression vector according to claim 3; (iv) The host cell according to claim 4; (v) The composition according to any one of claims 5 to 7.

10. A method for preparing a snake venom-like tripeptide, characterized in that: include: (a) mixing amino acids with the mutant 1 and the mutant 2 of the mutant according to claim 1 to obtain a snake venom-like tripeptide; or (b) mixing amino acids, Pro-Dab-Bn and the mutant 2 in the mutant according to claim 1 to obtain a snake venom-like tripeptide; or (c) mixing the amino acids and the fusion protein in the mutant according to claim 1 to obtain a snake venom-like tripeptide; or (d) expressing the nucleic acid molecule according to claim 2, and mixing the obtained protein product with amino acids to obtain a snake venom-like tripeptide; or (e) expressing the expression vector according to claim 3, and mixing the obtained protein product with amino acids to obtain a snake venom-like tripeptide; or (f) culturing the host cell according to claim 4, and mixing the obtained protein product with amino acids to obtain a snake venom-like tripeptide; or (g) mixing the amino acid and the composition according to any one of claims 5 to 7 to obtain a snake venom-like tripeptide; The amino acids are β-ala, L-Pro and / or Dab-Bn; The mutant 1 catalyzes L-Pro and Dab-Bn to synthesize Pro-Dab-Bn; The mutant 2 catalyzes β-ala and Pro-Dab-Bn to synthesize β-ala-Pro-Dab-Bn; The fusion protein of the mutant 1 and the mutant 2 catalyzes β-ala, L-Pro and Dab-Bn to synthesize β-ala-Pro-Dab-Bn.

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