Leucine ligase mutant and its application

Abstract

The present invention relates to the field of biotechnology, and particularly to leucine ligase mutants and their applications. The present invention provides ligase mutants, applications, and a method for preparing dipeptides. In the present invention, a mutant library is constructed by mutating specific amino acid sites of the enzyme; then, several mutants are found to have high catalytic activity towards Leu-AA through screening, and functional dipeptides such as Leu-Leu, Leu-Ile, and Leu-Val can be prepared using them.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to leucine ligase mutants and their applications. Background Art

[0002] Short peptides containing L-leucine are widely present in nature and have various uses; for example, L-leucine-L-isoleucine (Leu-Ile) has been reported to have a neuroprotective effect, L-leucine-L-leucine (Leu-Leu) is used as an antibody-drug conjugate (ADC-linker), and L-leucine-L-valine (Leu-Val) can be used as the molecular backbone of antimicrobial and antimalarial drugs, etc. So far, the large-scale production of such dipeptides is still through traditional chemical synthesis processes, and there are few reports on the method of using enzymes, especially amino acid ligases, to prepare such dipeptides. (References: James A. Ezugwu "Novel Leu-Val Based Dipeptide as Antimicrobial and Antimalarial Agents: Synthesis and Molecular Docking" 2020; Masayoshi Nakatani "Periocular injection of in situ hydrogels containing Leu-Ile, an inducer for neurotrophic factors, promotes retinal ganglion cell survival after optic nerve injury" 2011)

[0003] Traditional methods for preparing dipeptides:

[0004] 1) The most commonly used method for dipeptides on the market is chemical synthesis. In this preparation process, it is generally necessary to first selectively protect the functional groups that are not desired to participate in the reaction, then perform bimolecular chemical coupling, and finally deprotect. Therefore, the reaction steps are numerous and the overall yield is low; at the same time, racemates will inevitably be generated during the chemical coupling process. Since the racemate is very similar in properties to the product and cannot be effectively separated, the separation cost is greatly increased; therefore, chemical synthesis of dipeptides is not an optimal choice either economically or environmentally (chemical preparation requires a large amount of organic solvents).

[0005] 2) Many documents have also reported the use of amide bond hydrolases, such as proteases, aminopeptidases, etc., to carry out the reverse reaction in organic solvents to achieve the enzymatic preparation of dipeptides. Compared with the above chemical synthesis process, the two amino acid substrates do not need to be subjected to corresponding group protection, the reaction conditions are relatively mild, and no racemate is produced. However, this reaction is an equilibrium reaction, it is difficult to achieve high-yield conversion, and organic solvents also need to be used.

[0006] 3) A small number of documents have reported the use of amino acid ligases to directly link amino acids to generate the corresponding dipeptides. This method not only does not require the protection of amino acids, but also can achieve high-yield conversion under mild conditions. For example, common glutathione and carnosine. However, the amino acid ligases reported so far are very few in the production process of dipeptides, which is mainly due to the narrow catalytic substrates of this enzyme.

[0007] As mentioned above, the current industrial production method of Leu-AA dipeptide is mainly chemical synthesis, but this method has a long preparation route, a large amount of organic solvents used, a low final yield and poor product quality. Although some documents have reported the enzymatic preparation of some dipeptides, due to the substrate specificity of the enzyme catalyst itself, it cannot be used to prepare Leu-AA. Therefore, exploring and modifying amino acid ligases to prepare Leu-AA becomes of practical application value. Summary of the Invention

[0008] In view of this, the present invention provides a leucine ligase mutant and its application. The enzyme preparation method has many advantages, such as simple preparation, high yield, good product quality, good environmental compatibility, low carbon emission, easy to scale up production, etc.

[0009] To achieve the above invention purpose, the present invention provides the following technical solutions:

[0010] The present invention provides a mutant of amino acid ligase, which:

[0011] The amino acids at positions 14, 83, 84, 85, 231, 232, 235, 290, 292, 294 and 336 are mutated to aspartic acid, leucine, proline, glutamine, phenylalanine, threonine, alanine, threonine, tryptophan, arginine and glycine in sequence;

[0012] Or

[0013] The amino acids at positions 14, 83, 84, 85, 231, 232, 235, 290, 292, 294 and 336 are mutated to aspartic acid, glycine, leucine, arginine, methionine, alanine, phenylalanine, threonine, tryptophan, arginine and valine in sequence;

[0014] or

[0015] The amino acids at positions 14, 83, 84, 85, 231, 232, 235, 290, 292, 294 and 336 are sequentially mutated to glutamine, isoleucine, methionine, glutamic acid, phenylalanine, serine, alanine, threonine, tyrosine, lysine and alanine;

[0016] or

[0017] The amino acids at positions 14, 83, 84, 85, 231, 232, 235, 290, 292, 294 and 336 are sequentially mutated to glutamic acid, valine, aspartic acid, leucine, tyrosine, serine, alanine, threonine, valine, histidine and phenylalanine;

[0018] or

[0019] The amino acids at positions 14, 83, 84, 85, 231, 232, 235, 290, 292, 294 and 336 are sequentially mutated to threonine, valine, aspartic acid, isoleucine, threonine, lysine, serine, threonine, phenylalanine, glutamic acid and valine;

[0020] The amino acid ligase is derived from Pseudomonas syringae; and / or

[0021] The amino acid ligase includes one or more of the following:

[0022] (I) The amino acid ligase that links L-leucine and L-leucine;

[0023] (II) The amino acid ligase that links L-leucine and L-isoleucine;

[0024] (III) The amino acid ligase that links L-leucine and L-valine.

[0025] In some specific embodiments of the present invention, the above-mentioned mutant has:

[0026] (1) An amino acid sequence as shown in any one of SEQ ID NO.2 to SEQ ID NO.6; or

[0027] (2) An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence as shown in (1), and having the same or similar function as that in (1); or

[0028] (3) An amino acid sequence having at least 70% homology with the amino acid sequence as shown in (1) or (2);

[0029] The plurality is from 2 to 130.

[0030] The present invention also provides a nucleic acid molecule encoding the above mutant;

[0031] The nucleic acid molecule has:

[0032] (4), a nucleotide sequence as shown in any one of SEQ ID NO.8 to SEQ ID NO.12; or

[0033] (5), a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (4), and having the same or similar function as (4); or

[0034] (6), a nucleotide sequence having at least 70% homology with the nucleotide sequence as shown in (4) or (5);

[0035] The plurality is from 2 to 400.

[0036] The present invention also provides an expression vector, comprising the above nucleic acid molecule and an acceptable genetic element.

[0037] The present invention also provides a host cell, comprising the above nucleic acid molecule or the above expression vector.

[0038] The present invention also provides a composition, comprising the above mutant and adenosine triphosphate.

[0039] In some specific embodiments of the present invention, the above composition further comprises acetate kinase and acetyl phosphate.

[0040] In some specific embodiments of the present invention, for the above composition, the acetate kinase has:

[0041] (7), an amino acid sequence as shown in SEQ ID NO.1; or

[0042] (8), an amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence as shown in (7), and having the same or similar function as (7); or

[0043] (9), an amino acid sequence having at least 70% homology with the amino acid sequence as shown in (7) or (8);

[0044] The plurality is from 2 to 120.

[0045] The present invention also provides the use of any one of the following in the synthesis of dipeptides:

[0046] (I), the above mutant;

[0047] (II), the above nucleic acid molecule;

[0048] (III), the above expression vector;

[0049] (IV), the above host cell;

[0050] (V), the above composition;

[0051] The dipeptide includes one or more of L-leucine-L-leucine, L-leucine-L-isoleucine or L-leucine-L-valine.

[0052] The present invention also provides a method for preparing a dipeptide, including:

[0053] (a), mixing the above mutant with an amino acid to obtain a dipeptide; or

[0054] (b), expressing the above nucleic acid molecule, mixing the obtained protein product with an amino acid to obtain a dipeptide; or

[0055] (c), expressing the above expression vector, mixing the obtained protein product with an amino acid to obtain a dipeptide; or

[0056] (d), culturing the above host cell, mixing the obtained protein product with an amino acid to obtain a dipeptide; or

[0057] (e), mixing the above composition with an amino acid to obtain a dipeptide;

[0058] The dipeptide includes one or more of L-leucine-L-leucine, L-leucine-L-isoleucine or L-leucine-L-valine;

[0059] The amino acid includes L-leucine, L-isoleucine and / or L-valine.

[0060] The mutant of the present invention has the following effects:

[0061] The present invention utilizes the characteristic that the amino acid ligase in Pseudomonas syringae can catalyze a variety of amino acid substrates, and by mutating the amino acid residues in its catalytic active pocket, a dipeptide compound capable of catalyzing the production of L-leucine and L-leucine, L-isoleucine, L-valine is finally obtained. This method is much better than the traditional chemical dipeptide synthesis and the method of synthesizing dipeptides by the reverse reaction of proteolytic enzymes. It shows outstanding advantages in terms of production cost, energy consumption, product quality and green index. Therefore, the large-scale production of this method will be the best choice for the production of the above dipeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0063] Figure 1Showing the HPLC detection of the reaction process of leucine ligase LL1 catalyzing the formation of Leu-Leu dipeptide; among them, the upper figure shows Leu-Leu-LL1-0h; the lower figure shows Leu-Leu-LL1-3h;

[0064] Figure 2 Showing the NMR spectrum of Leu-Leu;

[0065] Figure 3 Showing the mass spectrum of Leu-Leu;

[0066] Figure 4 Showing the HPLC detection results of the reaction catalyzed by LI2 enzyme to generate Leu-Ile; among them, the upper figure shows 0h; the lower figure shows 3h;

[0067] Figure 5 Showing the HPLC detection results of the reaction catalyzed by LI3 enzyme to generate Leu-Ile; among them, the upper figure shows 0h; the lower figure shows 3h;

[0068] Figure 6 Showing the mass spectrum results of Leu-Ile dipeptide; its molecular weight is 245.4;

[0069] Figure 7 Showing the detection results of the 5-hour reaction process of LV4 and LV5 enzymes catalyzing the formation of Leu-Val; among them, the upper figure shows LV4; the lower figure shows LV5;

[0070] Figure 8 Showing the mass spectrum results of Leu-Val dipeptide; its molecular weight is 231.3;

[0071] Figure 9 Showing the gel electrophoresis diagram of the enzyme; among them, the left figure is the mutant enzyme; the right figure is the ackA enzyme. Detailed implementation mode

[0072] The present invention discloses a leucine ligase mutant and its application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0073] An amino acid ligase (Uniprot: Q842E2) in Pseudomonas syringae is reported to have a very broad substrate range (Reference: Toshinobu Arai, L-Amino acid ligase from Pseudomonas syringae producing tabtoxin can be used for the enzymatic synthesis of various functional peptides). In the present invention, a mutant library was constructed by mutating specific amino acid sites of this enzyme. Then, several mutants were found to have high catalytic activity towards Leu-AA (here Leu-AA refers to Leu-leu, Leu-Ile, Leu-Val, the same below), and they can be used to prepare three functional dipeptides, namely Leu-Leu, Leu-Ile, and Leu-Val.

[0074] The mutant library of the amino acid ligase in Pseudomonas syringae provided by the present invention makes it possible for the direct enzymatic preparation of Leu-AA.

[0075] The preparation route of the amino acid ligase method in the present invention:

[0076]

[0077]

[0078] This route uses cheap amino acids as raw materials, without the need for additional functional group protection, and is converted into dipeptide products with high yield under the action of equimolar adenosine triphosphate (ATP) and the corresponding amino acid ligase. The ATP in the reaction can also be further reduced in its usage amount by using a recycling regeneration system. Therefore, this preparation route is concise, has a high yield, good product quality (no racemate impurities are generated), has a high green index in production, and is easy to scale up production.

[0079] In the present invention, the corresponding amino acid ligase can directly convert amino acids into the corresponding dipeptide products in one step in a buffer. The adenosine triphosphate (ATP) required in the reaction can be added in an equimolar amount, or in a catalytic equivalent amount together with a regeneration system (acetate kinase ackA / acetyl phosphate).

[0080] Enzyme-related information:

[0081] Amino acid ligase (Ligase LL / LI / LV): derived from Pseudomonas syringae (Uniprot ID: Q842E2, EC 6.3.2.49);

[0082] Acetate kinase (ackA): derived from Escherichia coli (Uniprot ID: P0A6A3, EC 2.7.2.1).

[0083] The mutant amino acid and nucleotide sequences involved in the present invention are shown in Table 1 and Table 2 respectively:

[0084] Table 1

[0085]

[0086]

[0087] Table 2

[0088]

[0089]

[0090]

[0091]

[0092] Fermentation production of the enzyme:

[0093] All the enzymes required for the present invention are prepared by synthesizing the corresponding genes by the company, constructing them on a specific expression plasmid, and then producing them through fermentation of Escherichia coli. The specific steps are as follows: After optimizing the sequences of the genes corresponding to the above enzymes, they are ordered from General Biosystems (Chuzhou, Anhui) for synthesis. Then, NdeI / XhoI restriction enzyme sites are introduced and subcloned into the pET 28a expression vector. The plasmid with the correct sequence is transferred into E. coli (BL21) competent cells for plate culture (Qingke Biotechnology) and monoclonal small-scale liquid culture. The bacteria with correct protein expression are finally subjected to stepwise enlarged liquid culture. Specifically, a single colony is transferred into 5 mL of LB culture medium containing 50 μM kanamycin and cultured at 37 °C. When the cells grow to the logarithmic phase, they are inoculated into 250 mL of LB culture medium containing the same antibiotic. When they grow to the logarithmic phase again, they are transferred into a 5 L culture fermenter for culture and final protein expression. During the 5 L fermenter culture, when the cell OD reaches about 20, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) is added at 25 °C to induce protein expression for 6 hours. Finally, the cells are collected by high-speed centrifugation (4000 rpm, 20 min) to obtain 40 - 70 g of wet cells with overexpressed enzymes. Take a small amount of cells and first mix them evenly with Tris.HCl buffer (50 mM, pH 8.0) on an ice bath. Then, the cells are broken by the freeze-thaw method, and the cell walls are removed by high-speed centrifugation. The supernatant is run on an SDS-PAGE gel (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to determine protein expression. The bacterial cells with correct protein expression are used for the next catalytic experiment. Specifically, the remaining cells are mixed evenly with Tris.HCl buffer (50 mM, pH 8.0) at low temperature (mixed at about 10 g of wet cells: 200 mL of buffer), and then the cell walls are broken by low-temperature high-pressure. After removing the cell walls by high-speed centrifugation (16000 rpm, 45 min), the enzyme-containing supernatant is obtained for standby (the enzyme activity obtained is 100 - 450 U / mL, where U is the amount of enzyme required to convert 1 μmol of substrate at room temperature for one minute). The composition of the LB medium is: 1% tryptone, 0.5% yeast extract, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate, and 5% glycerol.

[0094] Unless otherwise specified, the raw materials, reagents, consumables, and instruments involved in the present invention are all ordinary commercially available products and can be purchased from the market.

[0095] The present invention will be further described below in conjunction with the examples:

[0096] Example 1: Preparation of L-leucine-L-leucine (Leu-Leu) using ligase (ligase LL1)

[0097]

[0098] 26.2 g of L-leucine (200 mM) and 55.5 g of adenosine monophosphate sodium salt (ATP, 105 mM) were added to 1 L of 100 mM Tris-HCl solution at pH 7.5. After adjusting the pH of the reaction system to 7.5 with an aqueous NaOH solution, 2500 U of ligase Ligase LL1 was added to initiate the reaction. The reaction system was gently stirred and the pH was maintained between 7.0 and 8.5. After 3 hours, HPLC detection showed that the raw materials had basically reacted completely. After the reaction, the pH of the reaction system was adjusted to 2.0 with an aqueous HCl solution to denature and precipitate the enzymes in the reaction system. Then, the protein solid was removed by centrifugation. After adjusting the pH of the reaction solution back to 7.0, it was directly loaded onto a D201 anion exchange resin for impurity removal. Finally, after desalting with a reverse osmosis membrane, it was concentrated and crystallized (ethanol:water, 2:1, v:v) to obtain 18.5 g of L-leucine-L-leucine (Leu-Leu) dipeptide (yield 76%). The reaction process was detected by HPLC, see Figure 1 。The final product, L-leucine-L-leucine (Leu-Leu) dipeptide, was confirmed by NMR and mass spectrometry. The results are shown in Figure 2 、 Figure 3 。The electrophoresis result of the enzyme LL1 used in the reaction is shown in Figure 9 。

[0099] Signal assignment of the NMR spectrum: 1 H NMR (400 MHz, Deuterium Oxide) δ 4.22 (dd, J = 9.5, 4.5 Hz, 1H), 4.06–4.01 (m, 1H), 1.78–1.72 (m, 2H), 1.65–1.61 (m, 2H), 0.96 (ddd, J = 22.0, 9.4, 5.7 Hz, 12H).

[0100] Example 2: Preparation of L-leucine-L-isoleucine Leu-Ile using ligase (ligase LI2 / LI3)

[0101]

[0102] Similar to the preparation of Leu-Leu above, 13.1 g of L-leucine (100 mM), 13.1 g of L-isoleucine (100 mM) and 55.5 g of adenosine 5'-triphosphate monosodium salt (ATP, 105 mM) were added to 1 L of 100 mM Tris.HCl solution at pH 7.5. Then, after adjusting the pH value of the reaction system to 7.5, ligase Ligase LI2 1800 U or Ligase LI3 2200 U was added to initiate the reaction. The reaction was slowly stirred at room temperature (25 °C) (150 rpm) and the pH of the system was maintained between 7.0 and 8.5 during the reaction. After 2 - 3 hours, when it was detected that the raw materials had completely reacted, the reaction was terminated by adjusting the pH to 2.0 with aqueous HCl solution and the enzyme was precipitated. After removing the enzyme by high-speed centrifugation (10,000 rpm), the pH of the reaction solution was adjusted to 7.0. Finally, D201 anion exchange resin was used to remove adenosine diphosphate and monophosphate impurities in the reaction system. The crude dipeptide obtained was first desalted by reverse osmosis membrane and then concentrated and crystallized (ethanol: water, 1:1 - 3:1, v:v). Finally, 19.3 - 21.2 g of L-leucine-L-isoleucine Leu-Ile dipeptide was obtained (yield 79 - 87%). The reaction process was detected by HPLC. The results of Leu-Ile dipeptide formation are shown in Figure 4 and Figure 5 . The product was sent for mass spectrometry confirmation. The results are shown in Figure 6 .

[0103] Example 3: Preparation of L-leucine-L-valine Leu-Val using ligase (ligase LV4 / LV5)

[0104]

[0105] Since these several enzymes are all mutated from a single parent, their overall properties are relatively similar. However, their activities in catalyzing different substrates and their stabilities during the catalytic process are slightly different. Therefore, the preparation of Leu-Val is similar to the above reaction, but there are slight differences in the amount of enzyme used and the reaction time. Add 19.6 g of L-leucine (150 mM), 17.6 g of L-valine (150 mM), and 83.3 g of adenosine monophosphate sodium salt (ATP, 157 mM) to 1 L of 100 mM Tris.HCl solution at pH 8.0. Then, after adjusting the pH value of the reaction system to 8.0, add 2500 U of ligase Ligase LV4 or 4000 U of Ligase LV5 to start the reaction. Stir slowly at room temperature (25 °C) (150 rpm) and maintain the pH of the system between 7.5 and 9.0 during the reaction. After 3 - 5 hours, when it is detected that the raw materials have completely reacted, adjust the pH to 2.0 with aqueous HCl solution to terminate the reaction and precipitate the enzyme. After centrifuging at high speed to remove the enzyme (10,000 rpm), adjust the pH of the reaction solution to 7.0. Finally, use D201 anion exchange resin to remove adenosine diphosphate and monophosphate impurities in the reaction system. The crude dipeptide obtained is first desalted using a reverse osmosis membrane and then concentrated and crystallized (ethanol:water, 1.5:1 - 2.5:1, v:v). Finally, 28.6 - 31.4 g of L-leucine-L-valine Leu-Val dipeptide is obtained (yield 86 - 91%). After 5 hours of reaction, HPLC detection is carried out to confirm the formation of the product dipeptide. The results are shown in Figure 7 . The purified product is sent for mass spectrometry confirmation. The results are shown in Figure 8 .

[0106] Example 4: Preparation of Leu-Val using ligase (ligase LV4) and an ATP regeneration system.

[0107]

[0108] All of the above conversions used an equivalent amount of adenosine triphosphate (ATP). In practical applications, we can also adopt an ATP recycling system, which can effectively reduce the dosage of ATP. Here, the preparation of Leu-Val using Ligase LV4 is taken as an example, and the preparation of other dipeptides is also applicable. Add 13.1 g of L-leucine (100 mM), 11.7 g of L-valine (100 mM), 2.6 g of adenosine triphosphate monosodium salt (ATP, 5 mM), and 100 mL of 1.2 N acetic acid phosphate solution (120 mM) to 1 L of 100 mM Tris.HCl solution with a pH of 7.5. After adjusting the pH of the reaction system to 7.5, add 3000 U of acetate kinase ackA and 1500 U of ligase Ligase LV4 to initiate the reaction. Stir slowly at 30 °C and maintain the pH of the reaction system between 7.0 and 8.5. After 2 hours, when it is detected that the raw materials have completely reacted, adjust the pH to 2.0 with an aqueous HCl solution to terminate the reaction and precipitate the enzyme. After centrifuging at high speed to remove the enzyme (10,000 rpm), adjust the pH of the reaction solution to 7.0. Finally, use D201 anion exchange resin to remove adenosine diphosphate and monophosphate impurities in the reaction system, use DEAE agarose gel resin to remove acetic acid, and finally collect the crude product and remove salt, concentrate and crystallize it using a reverse osmosis membrane (ethanol: water, 1.5:1 - 2.5:1, v:v). Finally, 17.3 - 19.5 g of L-leucine-L-valine Leu-Val dipeptide is obtained (yield 75 - 85%).

[0109] The preparation method of acetic acid phosphate (Acetyl phosphate) is attached:

[0110] Dissolve 135 mL of phosphoric acid (85%, 2.0 mol) in 1.2 L of ethyl acetate, and then cool it to 0 °C; slowly drop 376 mL of cooled acetic anhydride (4.0 mol) into this solution. Stir and react the above mixture at 0 °C for 6 hours, and pour it into a 5 L reaction flask containing 1 L of water, 500 g of ice, and 168 g of sodium bicarbonate. Continue to stir the mixture at low temperature until no bubbles are generated. After separating the upper ethyl acetate phase and discarding it, adjust the pH of the remaining aqueous phase to ~3 and extract it twice with 2.0 L and 1.0 L of ethyl acetate to remove most of the remaining acetic acid. Finally, adjust the pH of the aqueous solution containing acetic acid phosphate to neutral with sodium hydroxide for standby. It is proved by enzyme activity test that 1.5 L of 1.2 N acetic acid phosphate aqueous solution is obtained.

[0111] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A mutant of an amino acid ligase, characterized in that: Its amino acid sequence is shown in any one of SEQ ID NO.2 to SEQ ID NO.

6.

2. A nucleic acid molecule, characterized in that, Encoding the mutant as claimed in claim 1; The nucleotide sequence of the nucleic acid molecule is shown in any one of SEQ ID NO.8 to SEQ ID NO.

12.

3. Expression vector, characterized in that, Comprising the nucleic acid molecule as claimed in claim 2, and acceptable genetic elements.

4. A host cell, characterized in that, Comprising the nucleic acid molecule as claimed in claim 2 or the expression vector as claimed in claim 3.

5. A composition, characterized in that, Comprising the mutant as claimed in claim 1 and adenosine triphosphate.

6. The composition according to claim 5, wherein Also comprising acetate kinase and acetyl phosphate.

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

1.

8. Use of any one of the following in the synthesis of dipeptides: (I), the mutant as claimed in claim 1; (II), the nucleic acid molecule as claimed in claim 2; (III), the expression vector as claimed in claim 3; (IV), the host cell as claimed in claim 4; (V), the composition as claimed in any one of claims 5 to 7; The dipeptide comprises one or more of L-leucine-L-leucine, L-leucine-L-isoleucine or L-leucine-L-valine; Among them, Preparing L-leucine-L-leucine with the mutant having the amino acid sequence shown in SEQ ID NO.2; Preparing L-leucine-L-isoleucine with the mutant having the amino acid sequence shown in SEQ ID NO.3 or 4; Preparing L-leucine-L-valine with the mutant having the amino acid sequence shown in SEQ ID NO.5 or 6.

9. A method for preparing a dipeptide, characterized in that, Comprising: (a), mixing the mutant as claimed in claim 1 with amino acids to obtain a dipeptide; or (b), expressing the nucleic acid molecule as claimed in claim 2, mixing the obtained protein product with amino acids to obtain a dipeptide; or (c), expressing the expression vector as claimed in claim 3, mixing the obtained protein product with amino acids to obtain a dipeptide; or (d), culturing the host cell as claimed in claim 4, mixing the obtained protein product with amino acids to obtain a dipeptide; or (e), mixing the composition as claimed in any one of claims 5 to 7 with amino acids to obtain a dipeptide; The dipeptide comprises one or more of L-leucine-L-leucine, L-leucine-L-isoleucine or L-leucine-L-valine; The amino acids comprise L-leucine, L-isoleucine and / or L-valine; wherein, preparing L-leucine-L-leucine with the mutant having the amino acid sequence shown in SEQ ID NO.2; preparing L-leucine-L-isoleucine with the mutant having the amino acid sequence shown in SEQ ID NO.3 or 4; preparing L-leucine-L-valine with the mutant having the amino acid sequence shown in SEQ ID NO.5 or 6.

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