CN115820574A - Leucine ligase mutant and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a leucine ligase mutant and application thereof. The invention provides a ligase mutant, application and a preparation method of dipeptide. The invention constructs a mutant library by mutating the specific amino acid site of the enzyme; then, several mutants are found to have high catalytic activity to Leu-AA by screening, and can be used for preparing functional dipeptides such as Leu-Leu, leu-Ile, leu-Val and the like.

Description

Leucine ligase mutant and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a leucine ligase mutant and application thereof.

Background

L-leucine-containing short peptides are widely found in nature and have a wide variety of uses; for example, L-leucine-L-isoleucine (Leu-Ile) is reported to have neuroprotective effect, L-leucine-L-leucine (Leu-Leu) is used as an antibody drug conjugate (ADC-linker), and L-leucine-L-valine (Leu-Val) is used as a molecular scaffold of antimicrobial, antimalarial drugs, etc. Up to now, the mass production of such dipeptides is still performed by the conventional chemical synthesis process, and the enzymatic method, especially the method for preparing such dipeptides by using amino acid ligase, has been reported. ( Reference: james A. Ezugwu "Novel Leu-Val Based peptide as analytical and analytical Agents Synthesis and Molecular packaging" 2020; masayoshi Nakatani "periodical injection of in situ hydrogels relating to Leue Ile, an induced for neural factors, a protein specific variant cell specific antibiotic approach" 2011 )

The conventional preparation method of dipeptide:

1) The most common method for dipeptide on the market is a chemical synthesis method, and in the preparation process, a functional group which is not expected to participate in the reaction is generally required to be selectively protected, then two molecules are chemically coupled, and finally deprotection is carried out, so that the reaction steps are more, and the overall yield is low; meanwhile, raceme is inevitably generated in the chemical coupling process, and the raceme and a product have similar properties and cannot be effectively separated, so that the separation cost is greatly increased; therefore, chemical synthesis of dipeptides is not an optimal choice, either economically or environmentally (chemical preparation requires large amounts of organic solvents).

2) Many documents report the enzymatic preparation of dipeptides by reverse reaction in organic solvents using amide bond hydrolases such as proteolytic enzymes (proteases), aminopeptidases (aminopeptidases), etc., in contrast to the above-mentioned chemical synthesis processes; corresponding group protection is not needed for the two amino acid substrates, the reaction conditions are mild, and racemes cannot be generated; however, this reaction is an equilibrium reaction, and it is difficult to achieve a high yield conversion, and an organic solvent is also required.

3) A small number of documents report the use of amino acid ligase (aminoacid ligase) for the direct ligation of amino acids to form the corresponding dipeptides, which does not require protection of the amino acids and allows high yield conversion under mild conditions. Such as glutathione, carnosine, which are commonly used. However, the amino acid ligases reported so far are rare in the production of dipeptides, mainly due to the relatively narrow substrates that the enzymes catalyze.

As mentioned above, the current industrial production mode of Leu-AA dipeptide is mainly chemical synthesis, but the method has long preparation route, large organic solvent consumption, low final yield and poor product quality. Although some enzyme methods are reported in the literature to prepare certain dipeptides, the enzyme catalyst cannot be used for preparing Leu-AA due to the specificity of the enzyme catalyst to a substrate, and the research and modification of the amino acid ligase to prepare Leu-AA become practical application values.

Disclosure of Invention

In view of this, the invention provides the leucine ligase mutant and the application thereof, and the preparation method of the leucine ligase mutant has various advantages, such as simple preparation, high yield, good product quality, good environmental compatibility, low carbon emission, easy large-scale production and the like.

In order to achieve the above object, the present invention provides the following technical solutions:

the present invention provides mutants of amino acid ligase that:

amino acids 14, 83, 84, 85, 231, 232, 235, 290, 292, 294 and 336 are sequentially mutated into aspartic acid, leucine, proline, glutamine, phenylalanine, threonine, alanine, threonine, tryptophan, arginine and glycine;

or

Amino acids 14, 83, 84, 85, 231, 232, 235, 290, 292, 294 and 336 are sequentially mutated into aspartic acid, glycine, leucine, arginine, methionine, alanine, phenylalanine, threonine, tryptophan, arginine and valine;

or

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

or

Amino acids 14, 83, 84, 85, 231, 232, 235, 290, 292, 294 and 336 are mutated into glutamic acid, valine, aspartic acid, leucine, tyrosine, serine, alanine, threonine, valine, histidine and phenylalanine in sequence;

or

Amino acids 14, 83, 84, 85, 231, 232, 235, 290, 292, 294 and 336 are mutated into threonine, valine, aspartic acid, isoleucine, threonine, lysine, serine, threonine, phenylalanine, glutamic acid and valine in sequence;

the amino acid ligase is derived from pseudomonas syringae; and/or

The amino acid ligase comprises one or more of:

(I) The amino acid ligase which links L-leucine and L-leucine;

(II) the amino acid ligase that links L-leucine and L-isoleucine;

(III) the amino acid ligase which ligates L-leucine and L-valine.

In some embodiments of the invention, the above mutants have:

(1) An amino acid sequence shown in any one of SEQ ID NO. 2-SEQ ID NO. 6; or

(2) The amino acid sequence shown in the (1) is obtained by substituting, deleting or adding one or more residues, and the function is the same as or similar to that of the (1); or

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

the plurality is 2 to 130.

The invention also provides nucleic acid molecules encoding the above mutants;

the nucleic acid molecule has:

(4) A nucleotide sequence shown in any one of SEQ ID NO. 8-SEQ ID NO. 12; or

(5) The nucleotide sequence obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (4) has the same or similar functions as the nucleotide sequence shown in (4); or

(6) A nucleotide sequence having at least 70% homology with the nucleotide sequence shown in (4) or (5);

the plurality is 2 to 400.

The invention also provides an expression vector, which comprises the nucleic acid molecule and an acceptable gene element.

The invention also provides a host cell comprising the nucleic acid molecule or the expression vector.

The invention also provides a composition comprising the mutant and adenosine triphosphate.

In some embodiments of the present invention, the composition further comprises acetate kinase and acetate phosphate.

In some embodiments of the invention, the above composition, the acetate kinase has:

(7) And an amino acid sequence shown as SEQ ID NO. 1; or

(8) The amino acid sequence obtained by substituting, deleting or adding one or more residues in the amino acid sequence shown in the (7), and the function is the same as or similar to that of the (7); or

(9) An amino acid sequence having at least 70% homology with the amino acid sequence shown in (7) or (8);

the plurality is 2 to 120.

The invention also provides the application of any one of the following in dipeptide synthesis:

(I) The above mutant;

(II) the above-mentioned nucleic acid molecule;

(III) the above expression vector;

(IV) the above host cell;

(V) the above composition;

the dipeptide includes one or more of L-leucine-L-leucine, L-leucine-L-isoleucine, or L-leucine-L-valine.

The invention also provides a preparation method of the dipeptide, which comprises the following steps:

(a) Mixing the mutant with amino acid to obtain dipeptide; or

(b) Expressing the nucleic acid molecule, and mixing the obtained protein product with amino acid to obtain dipeptide; or

(c) Expressing the expression vector, and mixing the obtained protein product with amino acid to obtain dipeptide; or

(d) Culturing the host cell, and mixing the obtained protein product with amino acid to obtain dipeptide; or

(e) Mixing the above composition with an amino acid 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 include L-leucine, L-isoleucine and/or L-valine.

The mutant of the invention has the following effects:

the invention utilizes the characteristic that amino acid ligase in pseudomonas syringae can catalyze various amino acid substrates, and finally obtains the dipeptide compound capable of catalyzing and producing L-leucine, L-isoleucine and L-valine by mutating amino acid residues in a catalytic activity pocket of the pseudomonas syringae. The method is greatly superior to the traditional chemical dipeptide synthesis and the method for synthesizing the dipeptide by utilizing the reverse reaction of the proteolytic enzyme. The method has outstanding advantages in the aspects of production cost, energy consumption, product quality and green index. Therefore, the scale production of the method is the best choice for producing the dipeptide.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows HPLC detection of the reaction process of Leu-Leu dipeptide catalyzed by leucine ligase LL1 enzyme; wherein, leu-Leu-LL1-0h is shown in the figure; leu-Leu-LL1-3h;

FIG. 2 shows the Leu-Leu NMR spectrum;

FIG. 3 shows a Leu-Leu mass spectrum;

FIG. 4 shows the HPLC detection result of Leu-Ile generated by LI2 enzyme-catalyzed reaction; wherein, the upper diagram is 0h; lower scheme 3h;

FIG. 5 shows the HPLC detection result of Leu-Ile generated by LI3 enzyme-catalyzed reaction; wherein, the upper diagram is 0h; lower scheme 3h;

FIG. 6 shows the results of Leu-Ile dipeptide mass spectrometry; its molecular weight is 245.4;

FIG. 7 shows the results of 5-hour reaction between Leu-Val catalyzed by LV4 and LV5 enzymes; wherein, the upper diagram is LV4; the lower diagram LV5;

FIG. 8 shows the Leu-Val dipeptide mass spectra results; the molecular weight thereof is 231.3;

FIG. 9 shows a gel electrophoresis of an enzyme; wherein, the left figure is mutant enzyme; the right panel is ackA enzyme.

Detailed Description

The invention discloses leucine ligase mutants and application thereof, and can be realized by appropriately improving process parameters by taking the contents of the leucine ligase mutants as reference by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

An Amino acid ligase (Uniprot: Q842E 2) in Pseudomonas syringae (Pseudomonas syringae) which has been reported to have a very broad substrate for the ligase (reference: toshinobu Arai, L-Amino acid ligase from Pseudomonas syringae growing mutant can used for the enzymatic synthesis of variant functional peptides), and the present invention constructs a library of mutants by mutating specific Amino acid sites of the enzyme; then, several mutants are found to have high catalytic activity on Leu-AA (herein, leu-Leu, leu-Ile, leu-Val, the same applies below) by screening, and can be used for preparing three functional dipeptides of Leu-Leu, leu-Ile and Leu-Val.

The amino acid ligase mutant library in pseudomonas syringae provided by the invention provides possibility for the direct enzyme preparation of Leu-AA.

The preparation route of the amino acid ligase method of the invention is as follows:

the route utilizes inexpensive amino acids as raw materials, does not need additional functional group protection, and can be converted into dipeptide products with high yield under the action of equivalent Adenosine Triphosphate (ATP) and corresponding amino acid ligase. ATP in the reaction can be further reduced by adopting a cyclic regeneration system. Therefore, the preparation method has the advantages of simple preparation route, high yield, good product quality (no racemate impurity generation), high green index in production and easy large-scale production.

The invention utilizes corresponding amino acid ligase to directly link and convert amino acid into corresponding dipeptide products in a buffer solution by one step. The Adenosine Triphosphate (ATP) required for the reaction can be added in equivalent amount, or catalyst equivalent amount can be added to a regeneration system (acetate kinase ackA/acetate phosphate).

Information relating to the enzyme:

amino acid Ligase (Ligase LL/LI/LV): derived from Pseudomonas syringae (Pseudomonas syringae, unit ID: Q842E2, EC 6.3.2.49);

acetate kinase (ackA): derived from Escherichia coli (Escherichia coli, unit ID: P0A6A3, EC 2.7.2.1).

The amino acid and nucleotide sequences of the mutant related to the invention are respectively shown in tables 1 and 2:

TABLE 1

TABLE 2

And (3) fermentation production of enzyme:

the enzyme required by the invention is prepared by constructing a corresponding gene synthesized by a company on a specific expression plasmid and then fermenting and producing escherichia coli; the method specifically comprises the following steps: the genes corresponding to the above enzymes were subjected to sequence optimization, synthesized by general biology company (Chuzhou, anhui), introduced with NdeI/XhoI cleavage sites, and subcloned into pET 28a expression vector. Coli (BL 21) competent cells to perform plate culture (prokaryotes) and monoclonal small-volume liquid culture, and the bacteria with correct protein expression are finally subjected to progressive amplification liquid culture. The method specifically comprises the steps of transferring a single colony into 5mL of LB culture solution (37 ℃) containing 50 mu M kanamycin for culture, inoculating the single colony into 250mL of LB culture solution containing the same antibiotics after the cells grow to the logarithmic phase, transferring the single colony into a 5L culture fermentation tank for culture when the cells grow to the logarithmic phase, and finally expressing the protein. In 5L fermenter culture, 0.5mM isopropyl-beta-D-thiogalactopyranoside (IPTG) was added at OD-20 to induce protein expression for 6 hours at 25 ℃ and finally cells were collected by high speed centrifugation (4000rpm, 20min) to obtain 40-70 g of wet cells with enzyme overexpression. A small amount of cells are taken and mixed with tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl) buffer solution (50mM, pH 8.0) uniformly on an ice basin, then the cells are crushed by a freeze-thaw method, and clear liquid after cell walls are removed by high-speed centrifugation is subjected to SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to determine protein expression. The bacterial cells with correct protein expression are used for carrying out the next catalytic experiment, and specifically, the residual cells and Tris.HCl buffer (50mM, pH 8.0) are uniformly mixed at low temperature (mixed by wet cells: 200mL buffer), then the cell walls are crushed at low temperature and high pressure, and enzyme-containing clear liquid is obtained for standby after high-speed centrifugation (16000rpm, 45min) is carried out to remove the cell walls (the obtained enzyme activity is 100-450U/mL, and U is the enzyme amount required for converting 1 mu mol of substrate at room temperature for one minute). The LB medium is composed of: 1% tryptone, 0.5% yeast powder, 1% NaCl,1% dipotassium hydrogen phosphate and 5% glycerol.

Unless otherwise specified, the raw materials, reagents, consumables and instruments according to the present invention are all commercially available products, and are commercially available.

The invention is further illustrated by the following examples:

example 1: preparation of L-leucine-L-leucine Leu-Leu Using ligase (ligase LL 1)

26.2 g of L-leucine (200 mM), 55.5 g of adenosine monosodium triphosphate (ATP, 105 mM) were added to 1L of 100mM Tris-HCl solution (pH 7.5), and after the pH of the reaction system was adjusted to 7.5 by means of an aqueous NaOH solution, the reaction was started by adding Ligase LL1 2500U, stirring gently while maintaining the pH of the reaction system at 7.0 to 8.5, and after 3 hours, completion of the reaction was detected by means of HPLC. After the reaction, the pH was adjusted to 2.0 with an aqueous HCl solution to denature and precipitate the enzyme in the reaction system, then the protein solid was removed by centrifugation, the pH of the reaction solution was adjusted back to 7.0, and the reaction solution was directly subjected to purification by loading on D201 anion exchange resin, and finally subjected to salt removal by a reverse osmosis membrane, concentration, and crystallization (ethanol: water, 2, 1, v) to obtain 18.5 g of L-leucine-L-leucine (Leu-Leu) dipeptide (yield 76%). The course of the reaction was checked by HPLC, see FIG. 1. The final product L-leucine-L-leucine (Leu-Leu) dipeptide was confirmed by nuclear magnetic and mass spectrometry, and the results are shown in FIGS. 2 and 3, and the results of electrophoresis with the enzyme LL1 for the reaction are shown in FIG. 9.

Signal attribution of nuclear magnetic spectrum: ¹ H NMR(400MHz,Deuterium Oxide)δ4.22(dd,J＝9.5,4.5Hz,1H),4.06–4.01(m,1H),1.78–1.72(m,2H),1.65–1.61(m,2H),0.96(ddd,J＝22.0,9.4,5.7Hz,12H).

example 2: preparation of L-leucine-L-isoleucine Leu-Ile Using ligase (ligase LI2/LI 3)

Similarly to the above Leu-Leu preparation, adding 13.1 g L-leucine (100 mM), 13.1 g L-isoleucine (100 mM) and 55.5 g adenosine triphosphate monosodium salt (ATP, 105 mM) to 1L 100mM Tris-HCl solution at pH 7.5, adjusting the pH of the reaction system to 7.5, then adding Ligase Ligase LI2 1800U or Ligase LI3 2200U to start the reaction, slowly stirring at room temperature (25 ℃) for 150rpm and maintaining the system pH between 7.0-8.5 during the reaction, after detecting that the raw material reaction is complete, adjusting the pH to 2.0 with HCl aqueous solution to terminate the reaction and precipitate the enzyme, after removing the enzyme (10000 rpm) by high speed centrifugation, adjusting the pH of the reaction solution to 7.0, finally removing adenosine diphosphate and monophosphate impurities in the reaction system with D201 anion exchange resin, collecting the crude product, concentrating and crystallizing after removing salts with reverse osmosis membrane (ethanol: water, 8978 xzv: 893-78 v), finally obtaining yield of isoleucine-1-19 g L-19 g leucine (79%). The reaction process was checked by HPLC, and the Leu-Ile dipeptide production results are shown in FIGS. 4 and 5. The product was confirmed by mass spectrometry and the results are shown in FIG. 6.

Example 3: preparation of L-leucine-L-valine Leu-Val Using ligase (ligase LV4/LV 5)

Since these enzymes are all mutated from one parent and have similar overall properties, but the activities of catalyzing different substrates and the stability in the catalytic process are slightly different, the Leu-Val preparation is similar to the above reaction, but has slight difference in enzyme dosage and reaction time. Adding 19.6 g of L-leucine (150 mM), 17.6 g of L-valine (150 mM) and 83.3 g of adenosine triphosphate monosodium salt (ATP, 157 mM) into 1L of 100mM Tris hydrochloric acid (Tris HCl) solution with the pH of 8.0, then adjusting the pH value of the reaction system to 8.0, adding Ligase Ligase LV4 2500U or Ligase 5 4000U, slowly stirring (150 rpm) at room temperature (25 ℃) and maintaining the system pH between 7.5 and 9.0 in the reaction process, after detecting that the raw material reaction is completed, adjusting the pH to 2.0 by using HCl aqueous solution to terminate the reaction and precipitate the enzyme, after removing the enzyme (10000 rpm) by high-speed centrifugation, adjusting the pH of the reaction solution to 7.0, finally removing the adenosine diphosphate and monophosphate impurities in the reaction system by using D201 anion exchange resin, collecting the obtained dipeptide, firstly removing salts by using a reverse osmosis membrane, concentrating and crystallizing (ethanol: water, 1.5: 1-2.5 v, 1 v, 1.5 v, 1.6 v), and finally obtaining the yield of valine (Val-4-86-91.6L). After 5 hours of reaction, HPLC analysis confirmed the formation of the dipeptide product, and the results are shown in FIG. 7. The purified product was confirmed by mass spectrometry, and the results are shown in FIG. 8.

Example 4: leu-Val was prepared using ligase (ligase LV 4) and the ATP regeneration system.

In the above conversion, adenosine Triphosphate (ATP) with equivalent weight is used, and in practical application, an ATP cyclic regeneration system can also be adopted, so that the use amount of ATP can be effectively reduced, and the preparation of Leu-Val by using Ligase LV4 is taken as an example, and the preparation of other dipeptides is also applicable. To 1L of 100mM Tris-HCl solution (pH 7.5) was added 13.1 g L-leucine (100 mM), 11.7 g L-valine (100 mM) and 2.6 g adenosine triphosphate monosodium salt (ATP, 5 mM), 100mL of 1.2N phosphate acetate solution (120 mM). Adjusting the pH value of a reaction system to 7.5, then respectively adding acetic kinase ackA 3000U and Ligase Ligase LV 4U to start reaction, slowly stirring at 30 ℃ and maintaining the pH value in the reaction system between 7.0-8.5, after 2 hours, detecting that the raw materials are completely reacted, adjusting the pH value to 2.0 by using HCl aqueous solution to terminate the reaction and precipitate the enzyme, removing the enzyme by high-speed centrifugation (10000 rpm), then adjusting the pH value of the reaction solution to 7.0, finally removing adenosine diphosphate and monophosphate impurities in the reaction system by using D201 anion exchange resin, removing acetic acid by using DEAE sepharose resin, finally collecting the obtained crude product, desalting, concentrating and crystallizing by using a reverse osmosis membrane (ethanol: water, 1.5.

Preparation method of acetic acid phosphate (Acetyl phosphate):

135mL of phosphoric acid (85%, 2.0 mol) was dissolved in 1.2L of ethyl acetate and then cooled to 0 ℃; to this solution 376mL of cooled acetic anhydride (4.0 mol) was slowly added dropwise. The mixture was stirred at 0 ℃ for 6 hours and poured into a 5l reaction flask containing 1l water, 500 g ice and 168 g sodium bicarbonate. The mixture was continued to be stirred at low temperature until no bubbles were generated. The upper layer of ethyl acetate was discarded after phase separation, and the remaining aqueous phase was adjusted to pH 3 and extracted twice with 2.0L, 1.0L of ethyl acetate to remove most of the remaining acetic acid. And finally, regulating the pH value of the aqueous solution containing the acetic acid phosphoric acid to be neutral by using sodium hydroxide for later use, and obtaining the 1.5L 1.2N acetic acid phosphoric acid aqueous solution through enzyme activity test.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A mutant of an amino acid ligase, characterized in that:

amino acids 14, 83, 84, 85, 231, 232, 235, 290, 292, 294 and 336 are sequentially mutated into aspartic acid, leucine, proline, glutamine, phenylalanine, threonine, alanine, threonine, tryptophan, arginine and glycine;

or

Amino acids 14, 83, 84, 85, 231, 232, 235, 290, 292, 294 and 336 are sequentially mutated into aspartic acid, glycine, leucine, arginine, methionine, alanine, phenylalanine, threonine, tryptophan, arginine and valine;

or

Amino acids 14, 83, 84, 85, 231, 232, 235, 290, 292, 294 and 336 are sequentially mutated into glutamine, isoleucine, methionine, glutamic acid, phenylalanine, serine, alanine, threonine, tyrosine, lysine and alanine;

or

Amino acids 14, 83, 84, 85, 231, 232, 235, 290, 292, 294 and 336 are sequentially mutated into glutamic acid, valine, aspartic acid, leucine, tyrosine, serine, alanine, threonine, valine, histidine and phenylalanine;

or

Amino acids 14, 83, 84, 85, 231, 232, 235, 290, 292, 294 and 336 are mutated into threonine, valine, aspartic acid, isoleucine, threonine, lysine, serine, threonine, phenylalanine, glutamic acid and valine in sequence;

the amino acid ligase is derived from pseudomonas syringae; and/or

The amino acid ligase comprises one or more of the following:

(I) The amino acid ligase which links L-leucine and L-leucine;

(II) the amino acid ligase that links L-leucine and L-isoleucine;

(III) the amino acid ligase which ligates L-leucine and L-valine.

2. The mutant of claim 1, having:

(1) An amino acid sequence shown in any one of SEQ ID NO. 2-SEQ ID NO. 6; or

(2) The amino acid sequence shown in the (1) is obtained by substituting, deleting or adding one or more residues, and the function is the same as or similar to that of the (1); or

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

the plurality is 2 to 130.

3. A nucleic acid molecule encoding the mutant of any one of claims 1 or 2;

the nucleic acid molecule has:

(4) A nucleotide sequence shown in any one of SEQ ID NO. 8-SEQ ID NO. 12; or

(5) The nucleotide sequence obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in the (4) has the same or similar functions as the nucleotide sequence in the (4); or

(6) A nucleotide sequence having at least 70% homology with the nucleotide sequence shown in (4) or (5);

the plurality is 2 to 400.

4. An expression vector comprising the nucleic acid molecule of claim 3, and an acceptable genetic element.

5. A host cell comprising the nucleic acid molecule of claim 3 or the expression vector of claim 4.

6. A composition comprising the mutant of any one of claims 1 or 2 and adenosine triphosphate.

7. The composition of claim 6, further comprising an acetate kinase and an acetate phosphate.

8. The composition of claim 6 or 7, wherein the acetate kinase has:

(7) And an amino acid sequence shown as SEQ ID NO. 1; or

(8) The amino acid sequence obtained by substituting, deleting or adding one or more residues in the amino acid sequence shown in the (7), and the function is the same as or similar to that of the (7); or

(9) An amino acid sequence having at least 70% homology with the amino acid sequence shown in (7) or (8);

the plurality is 2 to 120.

9. Use of any of the following in the synthesis of a dipeptide:

(I) The mutant of claim 1 or 2;

(II) the nucleic acid molecule of claim 3;

(III) the expression vector of claim 4;

(IV) the host cell of claim 5;

(V) a composition according to any one of claims 6 to 8;

the dipeptide includes one or more of L-leucine-L-leucine, L-leucine-L-isoleucine, or L-leucine-L-valine.

10. A process for producing a dipeptide, comprising:

(a) Mixing the mutant of claim 1 or 2 with an amino acid to obtain a dipeptide; or

(b) Expressing the nucleic acid molecule of claim 3, and mixing the protein product obtained with an amino acid to obtain a dipeptide; or

(c) Expressing the expression vector of claim 4, mixing the obtained protein product with an amino acid to obtain a dipeptide; or

(d) Culturing the host cell of claim 5 and mixing the protein product obtained with an amino acid to obtain a dipeptide; or

(e) Mixing a composition according to any one of claims 6 to 8 with an amino acid 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 include L-leucine, L-isoleucine and/or L-valine.

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