Application of mutation site as target spot in preparation of Arg-Arg dipeptide
By designing mutation sites in BsRizA enzyme and introducing a cheap ATP regeneration system, the problem of insufficient concentration and conversion rate of Arg-Arg dipeptide preparation products in the prior art was solved, and efficient and low-cost Arg-Arg dipeptide preparation was achieved.
Patent Information
- Application Number
- CN202510311231.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-14
- Publication Date
- 2025-06-06
AI Technical Summary
In the prior art, when preparing Arg-Arg dipeptide, the product concentration and conversion rate fail to meet the requirements of industrialization.
Through the design of mutation sites, high-performance BsRizA mutants were obtained, and an inexpensive ATP regeneration system was introduced to develop an Arg-Arg dipeptidase synthesis process that meets industrialization requirements.
The high concentration preparation of Arg-Arg dipeptide was achieved, and the product concentration was increased by more than 4 times, reducing material costs and improving conversion rate.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of bioengineering, and in particular to the application of mutation sites as targets in the preparation of Arg-Arg dipeptides. Background Art
[0002] TAT peptide (Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg) is a cell-penetrating peptide (CPPs) derived from HIV-1 virus, which can deliver peptides, proteins, oligonucleotides, etc. into cells under in vitro and in vivo conditions. Inspired by natural cell-penetrating peptides such as TAT peptide, artificially designed polyarginine peptides composed of 6-9 Args have been verified to have similar internalization functions. The chemical synthesis of these arginine-containing peptides requires the use of the costly Fmoc-Arg(Pbf)-OH to protect the side chain guanidine group, while the enzymatic synthesis of dipeptides and tripeptides such as Arg-Arg and Arg-Arg-Arg is an effective means to reduce costs. Arg-Arg can also be used to synthesize acetyl hexapeptide-8 (Ac-Glu-Glu-Met-Gln-Arg-Arg-NH 2 ).
[0003] However, the product concentration and conversion rate of the existing technology cannot meet the requirements of industrialization. Summary of the invention
[0004] In view of this, the present invention provides the application of mutation sites as targets in the preparation of Arg-Arg dipeptides. The present invention obtains high-performance BsRizA mutants, introduces a cheap ATP (adenosine triphosphate) regeneration system, and develops an enzymatic synthesis process of Arg-Arg dipeptides that meets the requirements of industrial green, low-cost, and high-concentration.
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides the use of mutation sites as targets in the preparation of Arg-Arg dipeptides; The mutation sites include: one or more of the 7th, 33rd and 35th positions of the amino acid sequence of the wild-type BsRizA enzyme.
[0006] The present invention also provides the use of the mutation site as a target in the preparation of Arg-Arg dipeptide; The mutation sites include: the 7th, 33rd and 35th positions of the amino acid sequence of the wild-type BsRizA enzyme.
[0007] The present invention also provides a mutant, which, based on the wild-type BsRizA enzyme, has one or more of the following amino acid site mutations: position 7, position 33 and position 35; the amino acid sequence of the wild-type BsRizA enzyme is shown in SEQ ID NO:1.
[0008] In some embodiments of the present invention, in the mutant, the sequence of SEQ ID NO: 1 is: MLRILLINSDKPEPIQFFQKDKETNDSINISVITRSCYAPLYSHWADHVYIVDDVTDLTVMKSLMLEILKVGPFDHIVSTTEKSILTGGFLRSYFGIAGPGFETALYMTNKLAMKTKLKMEGIPVADFLCVSQVEDIPAAGEKLGWPIIVKPALGSGALNTFIIHSLDHYEDLYSTSGGLGELKKNNSLMIAEKCIEMEEFH CDTLYADGEILFVSISKYTVPLLKGMAKIQGSFILSQNDPVYAEILELQKSVAQAFRITDGPGHLEIYRTHSGELIVGEIAMRIGGGGISRMIEKKFNISLWESSL NISVYRDPNLTVNPIEGTVGYFSLPCRNGTIKEFTPIEEWEKLAGILEVELLYQEGDVVDEKQSSSFDLARLYFCLENENEVQHLLALVKQTYYLHLTEDHMMNQ.
[0009]
[0010] In some embodiments of the present invention, in the above mutants, the mutation at position 7 includes: I7S or I7T; The mutation at position 33 includes: I33S or I33T; The mutation at position 35 includes: R35D or R35E.
[0011] In some embodiments of the present invention, the mutant described above has: (1), the amino acid sequence shown in SEQ ID NO: 2; or (2) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).
[0012] In some embodiments of the present invention, in the mutant, the sequence of SEQ ID NO: 2 is: MLRILLTNSDKPEPIQFFQKDKETNDSINISVTTESCYAPLYSHWADHVYIVDDVTDLTVMKSLMLEILKVGPFDHIVSTTEKSILTGGFLRSYFGIAGPGFETALYMTNKLAMKTKLKMEGIPVADFLCVSQVEDIPAAGEKLGWPIIVKPALGSGALNTFIIHSLDHYEDLYSTSGGLGELKKNNSLMIAEKCIEMEEFH CDTLYADGEILFVSISKYTVPLLKGMAKIQGSFILSQNDPVYAEILELQKSVAQAFRITDGPGHLEIYRTHSGELIVGEIAMRIGGGGISRMIEKKFNISLWESSL NISVYRDPNLTVNPIEGTVGYFSLPCRNGTIKEFTPIEEWEKLAGILEVELLYQEGDVVDEKQSSSFDLARLYFCLENENEVQHLLALVKQTYYLHLTEDHMMNQ.
[0013] The present invention also provides a nucleic acid molecule encoding the mutant.
[0014] In some embodiments of the present invention, the above-mentioned nucleic acid molecule has: (4) the nucleotide sequence shown in SEQ ID NO: 3; or (5) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence described in (4); or (6) a sequence having at least 80% homology with the nucleotide sequence described in (4) or (5); or (7) A complementary sequence of the nucleotide sequence described in (4), (5) or (6).
[0015]
[0016] The present invention also provides an expression vector, comprising: the above nucleic acid molecule.
[0017] The present invention also provides a host for transforming and / or transfecting the above expression vector.
[0018] The present invention also provides an enzyme composition, comprising the mutant and an auxiliary enzyme; the auxiliary enzyme comprises: polyphosphate kinase SlPPK2.
[0019] In some embodiments of the present invention, in the above enzyme composition, the amino acid sequence of the polyphosphate kinase S1PPK2 is as shown in SEQ ID NO:4: MKKNIYKKELYKLQVELVKFQKYVIEENVAVCLVLEGRDTAGKDGTIKRFTEHLSPREARTVALGVPSDKEKKSWYFQRYVPHLPSAGEIVFFNRSWYNRAGVEKVMGFCTKKQYKAFMEEVGSFEQMLTHSNIRFFKYYLDITKKEQKKRLEARKTDPLKQWKLSPIDAKAQKMWDAYSKARDDMFNKTSFIYAPWYVVHTDDKKEARINIMKHFLSLNDYPDKDKALLVYDHDVICKFDPVCYEKEMIAP.
[0020] In some embodiments of the present invention, in the above enzyme composition, the nucleotide sequence of the nucleic acid molecule encoding the polyphosphate kinase S1PPK2 is shown in SEQ ID NO: 5:.
[0021] The present invention also provides a primer set having sequences shown in SEQ ID NO:7 to SEQ ID NO:18.
[0022] The present invention also provides the use of the mutant, the nucleic acid molecule, the expression vector, the host and / or the enzyme composition in the preparation of Arg-Arg dipeptide.
[0023] The present invention also provides a method for preparing Arg-Arg dipeptide, which comprises mixing a raw material with the mutant or the enzyme composition to obtain Arg-Arg dipeptide; The raw materials include: arginine, magnesium chloride, sodium hexametaphosphate and disodium adenosine triphosphate.
[0024] In some embodiments of the present invention, in the above preparation method, the pH value of the mixture is 7.0-9.5.
[0025] In some embodiments of the present invention, in the above preparation method, the pH value of the mixture is 8.5.
[0026] In some embodiments of the present invention, in the above preparation method, the mixing temperature is 25-45°C.
[0027] In some embodiments of the present invention, in the above preparation method, the mixing temperature is 38°C.
[0028] In some embodiments of the present invention, in the above preparation method, the concentration of magnesium chloride is 20-80 mM.
[0029] In some embodiments of the present invention, in the above preparation method, the concentration of magnesium chloride is 60 mM.
[0030] In some embodiments of the present invention, in the above preparation method, the enzyme activity of the mutant is 173 U / mL; the enzyme activity of the auxiliary enzyme is 360 U / mL.
[0031] The rationally designed amino acid ligase BsRizA-I7T_I33T_R35E provided by the present invention has a product concentration increased by more than 4 times compared with the wild type; the ATP regeneration system composed of sodium hexametaphosphate and polyphosphate kinase reduces the use of the relatively expensive adenosine triphosphate disodium salt by 98%, thereby reducing the material cost; and the product concentration and conversion rate are further improved by optimizing the reaction parameters such as pH, temperature, and magnesium chloride concentration. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.
[0033] Figure 1 The optimal model for docking of BsRizA enzyme with Arg-Arg molecule is shown (protein is shown as surface potential, ligand is shown as stick); Figure 2 The hydrophobic pocket of the BsRizA enzyme that binds to Arg-Arg is shown (the hydrophobic region of the protein is shown in orange, and the hydrophilic region is shown in blue); Figure 3The modified sites Ile7, Ile33 and Arg35 that interact with Arg-Arg are shown; Figure 4 Shows the screening results of BsRizA mutants; Figure 5 The reaction formula for the synthesis of Arg-Arg by BsRizA-I7T_I33T_R35E enzyme and SlPPK2 enzyme is shown; Figure 6 Electrophoresis results of the fermentation-expressed BsRizA-I7T_I33T_R35E protein; Figure 7 The results of pH optimization of the Arg-Arg synthesis reaction of BsRizA-I7T_I33T_R35E enzyme and SlPPK2 enzyme are shown; Figure 8 The results of optimization of the reaction temperature of Arg-Arg synthesis by BsRizA-I7T_I33T_R35E enzyme and SlPPK2 enzyme are shown; Fig. 9 The results show the optimization of magnesium chloride concentration in the Arg-Arg synthesis reaction of BsRizA-I7T_I33T_R35E enzyme and SlPPK2 enzyme; Fig.10 Shows the laboratory amplification results of the synthesis of Arg-Arg by BsRizA-I7T_I33T_R35E enzyme and SlPPK2 enzyme. DETAILED DESCRIPTION
[0034] The invention discloses the application of a mutation site as a target in the preparation of an Arg-Arg dipeptide.
[0035] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.
[0036] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.
[0037] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.
[0038] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.
[0039] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.
[0040] The present invention firstly changes the hydrophobicity and surface charge of the BsRizA enzyme through rational design, thereby improving its binding ability and catalytic performance to the substrate Arg, thereby improving the conversion rate of the reaction.
[0041] Secondly, by introducing cheap sodium hexametaphosphate as a phosphate donor, the more expensive ATP is regenerated under the action of polyphosphate kinase SlPPK2, which greatly reduces the use of adenosine triphosphate disodium salt and thus reduces material costs.
[0042] Finally, the reaction conversion rate was further improved by optimizing the reaction pH, temperature and magnesium chloride concentration.
[0043] In Examples 1 to 3 of the present invention, all the raw materials and reagents used can be purchased from the market.
[0044] The present invention will be further described below in conjunction with embodiments: Example 1 Substrate Adaptation of BsRizA Enzyme The amino acid sequence of the amino acid ligase BsRizA from Bacillus subtilis NBRC3134 was input into the IDT codon optimization online tool https: / / sg.idtdna.com / CodonOpt to obtain the optimized nucleotide sequence, which was submitted to Beijing Qingke Biotechnology for whole gene synthesis and constructed on the pET-28a vector to obtain the expression plasmid pET28a-BsRizA.
[0045] Using BsRizA enzyme (PDB: 4WD3) as the target and Arg-Arg dipeptide as the ligand, molecular docking was performed using AutoDock Vina (e.g. Figure 1 As shown in the figure, the second Arg of the ligand is trapped in a hydrophobic pocket (as shown in the figure). Figure 2). Among the residues that interact with the ligand in this hydrophobic pocket, Figure 3 The hydrophobic Ile 7 and Ile 33 and the positively charged Arg 35 were selected as the modification sites. The primers shown in Table 1 were designed, and the plasmids of different mutants were constructed using the QuickChange site-directed mutagenesis kit (Agilent) with pET28a-BsRizA as a template.
[0046] Table 1
[0047]
[0048] The obtained plasmids were transformed into BL21 (DE3) competent cells (Shanghai Weidi Biotechnology), and then cultured in LB liquid medium (Kan) for two stages, and induced for expression at 20°C with 0.1mM IPTG for 16-20h. The cells were collected by centrifugation, and the disruption buffer (20mM Na 2 HPO 4 / NaH 2 PO 4 , 100 mM NaCl, pH 7.6) and then ultrasonically or high-pressure homogenized to obtain a crude enzyme solution (the bacteria of other enzymes not mentioned here can obtain the corresponding crude enzyme solutions according to the operation here).
[0049] The crude enzyme solution after ultrasonic disruption was diluted 10 times with disruption buffer, and 100 µL was added to 10 mL of Arg-Arg activation solution (Arg 20 mM, ATP-Na 2 15 mM MgCl 2 20 mM, pH 9), reacted at 38°C for 10 min, and the amount of product Arg-Arg generated was detected by high performance liquid chromatography (HPLC). The enzyme activity was calculated according to the following formula. The enzyme activity results of each mutant are shown in Table 2.
[0050]
[0051] Table 2
[0052]
[0053] In order to further observe the catalytic performance of each mutant, a longer reaction verification was carried out. Weigh 5.51g of adenosine triphosphate disodium salt (100mM, total reaction volume 100mL), 3.48g of L-Arg (200mM), and 0.61g of magnesium chloride hexahydrate (30mM) and dissolve them in 80mL of pure water, adjust pH 8.5 and make up to 90mL. Divide into 10 portions, preheat at 38℃, add 1mL of crude enzyme solution of different mutants respectively, and start the reaction. Samples were taken during the reaction, and the formation of product Arg-Arg was detected by high performance liquid chromatography (HPLC).
[0054] As shown in Table 3 and Figure 4 As shown in the figure, mutations of Ile at position 7 and Ile at position 33 to hydrophilic Ser or Thr can increase the amount of Arg-Arg generated. Mutation of Arg at position 35 to negatively charged Asp or Glu may also significantly increase the amount of product generated because of the formation of a new salt bridge with Arg at the second position of Arg-Arg. The optimal mutant is I7T_I33T_R35E, which can generate 56.4mM of product after 4h of reaction.
[0055] Table 3
[0056]
[0057] Example 2 Optimization of reaction conditions for the synthesis of Arg-Arg by BsRizA-I7T_I33T_R35E 1. Optimization of reaction pH In order to reduce the usage of the relatively expensive adenosine triphosphate disodium salt, an ATP regeneration system consisting of sodium hexametaphosphate and polyphosphokinase was introduced. The reaction formula is shown in Figure 5 The optimized nucleotide sequence of the polyphosphate kinase SlPPK2 from Sulfurovum lithotrophicum was submitted to Beijing Qingke Biotechnology for full gene synthesis and constructed on the pET-28a vector to obtain the expression plasmid pET28a-SlPPK2. The obtained plasmid was also transformed into BL21 (DE3) competent cells.
[0058] Take BsRizA-I7T_I33T_R35E BL21(DE3) and SlPPK2 BL21(DE3) glycerol bacteria and expand them in LB liquid medium (Kan) and inoculate them into small tanks for fermentation. The fermentation medium formula is as follows: Table 4
[0059]
[0060] When the biomass OD 600=40-60, start cooling to 20°C, then add 0.1mM IPTG to induce protein expression for 20-24h, and place the tank with OD 600 =160-240. The cells were collected by centrifugation and stored at -20°C for later use. Protein electrophoresis showed that BsRizA-I7T_I33T_R35E and SlPPK2 were expressed in the supernatant ( Figure 6 ).
[0061] Weigh 17.42g L-Arg (200mM, total reaction volume 500mL), 9.18g sodium hexametaphosphate (30mM), 3.05g magnesium chloride hexahydrate (30mM), 0.51g adenosine triphosphate disodium salt (2mM) and dissolve in 400mL pure water, divide into 5 portions, adjust pH to 7.0, 8.0, 8.5, 9.0, 9.5 respectively and make up to 89mL, preheat at 38℃ and add 10mL BsRizA-I7T_I33T_R35E crude enzyme solution (enzyme activity 173U / mL, the same below) and 1mL SlPPK2 crude enzyme solution (enzyme activity 360U / mL, the same below) to start the reaction. Samples were taken during the reaction, and the formation of the product Arg-Arg was detected by high performance liquid chromatography (HPLC).
[0062] As shown in Table 5 and Figure 7 As shown, although the pH 9.0 group produced the highest Arg-Arg 60.82 mM after 4 hours of reaction, the product concentration decreased too quickly from pH 9.0 to pH 9.5, so pH 8.5 was still selected as the optimal reaction pH.
[0063] Table 5
[0064]
[0065] 2. Optimization of reaction temperature Weigh 13.94g L-Arg (200mM, total reaction volume 400mL), 7.34g sodium hexametaphosphate (30mM), 2.40g magnesium chloride hexahydrate (30mM), 0.44g adenosine triphosphate disodium salt (2mM) and dissolve in 320mL pure water, adjust pH to 8.5 and make up to 356mL. Divide into 4 portions, preheat at 25℃, 32℃, 38℃, and 45℃, add 10mL BsRizA-I7T_I33T_R35E crude enzyme solution and 1mL SlPPK2 crude enzyme solution, and start the reaction.
[0066] As shown in Table 6 and Figure 8 As shown, the concentration of Arg-Arg generated in the 45℃ group after 1h reaction was the highest, but as time went on, the product generated in the 38℃ group after 4h reaction was the highest at 60.04 mM, which may be due to the poor stability of the enzyme at 45℃ and its rapid inactivation. 38℃ was still selected as the optimal reaction temperature.
[0067] Table 6
[0068]
[0069] 3. Optimization of magnesium chloride concentration Weigh 13.94g L-Arg (200mM, total reaction volume 400mL), 7.34g sodium hexametaphosphate (30mM), 0.44g adenosine triphosphate disodium salt (2mM) and dissolve in 320mL pure water, divide into 4 parts, weigh 0.41g magnesium chloride hexahydrate (20mM, reaction volume 100mL), 0.82g (40mM), 1.22g (60mM), 1.63g (80mM) respectively and dissolve, adjust pH 8.5 and make the volume to 89mL, preheat at 38℃ and add 10mL BsRizA-I7T_I33T_R35E crude enzyme solution and 1mL SlPPK2 crude enzyme solution to start the reaction. Samples were taken during the reaction, and the formation of the product Arg-Arg was detected by high performance liquid chromatography (HPLC).
[0070] As shown in Table 7 and Fig. 9 As shown in the figure, with the increase of magnesium chloride concentration, the Arg-Arg generated in the reaction for 4 hours also gradually increased, and the product generated in the 60mM group was 87.01mM, which was the highest. When it continued to increase to 80mM, the product concentration decreased, which may be caused by excessive metal ion toxicity. The optimal magnesium chloride concentration is 60mM.
[0071] Table 7
[0072]
[0073] Example 3 Laboratory Scale-up of Arg-Arg Synthesis by BsRizA-I7T_I33T_R35E Weigh 34.84g L-Arg (200mM, reaction volume 1000mL), 18.35g sodium hexametaphosphate (55mM), 12.20g magnesium chloride hexahydrate (60mM), 1.10g adenosine triphosphate disodium salt (2mM) and dissolve in 800mL pure water, adjust pH to 8.5 with ammonia water and dilute to 890mL, preheat at 38℃, add 100mL BsRizA-I7T_I33T_R35E crude enzyme solution and 10mL SlPPK2 crude enzyme solution to start the reaction. Samples were taken during the reaction, and the formation of the product Arg-Arg was detected by high performance liquid chromatography (HPLC).
[0074] like Fig.10 As shown, the reaction time was 4h to generate 87.03 mM Arg-Arg and the conversion rate was 87%, which was significantly higher than the previously reported generation of 10.5 mM Arg-Arg and 35% conversion rate.
[0075] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. Application of mutation sites as targets in the preparation of Arg-Arg dipeptides; The mutation sites include: One or more of positions 7, 33 and 35 of the amino acid sequence of the wild-type BsRizA enzyme.
2. A mutant, characterized in that Based on the wild-type BsRizA enzyme, it has one or more of the following amino acid site mutations: position 7, position 33 and position 35; the amino acid sequence of the wild-type BsRizA enzyme is shown in SEQ ID NO:
1.
3. The mutant according to claim 2, characterized in that The mutation at position 7 includes: I7S or I7T; The mutation at position 33 includes: I33S or I33T; The mutation at position 35 includes: R35D or R35E.
4. The mutant according to claim 2 or 3, characterized in that It has: (1), the amino acid sequence shown in SEQ ID NO: 2; or (2) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).
5. A nucleic acid molecule encoding the mutant according to any one of claims 2 to 4.
6. The nucleic acid molecule according to claim 5, characterized in that It has: (4) the nucleotide sequence shown in SEQ ID NO: 3; or (5) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence described in (4); or (6) a sequence having at least 80% homology with the nucleotide sequence described in (4) or (5); or (7) A complementary sequence of the nucleotide sequence described in (4), (5) or (6).
7. An expression vector, characterized in that include: The nucleic acid molecule according to claim 5 or 6.
8. A host, characterized in that Transformation and / or transfection with the expression vector according to claim 7.
9. An enzyme composition, characterized in that It comprises the mutant according to any one of claims 2 to 4 and an auxiliary enzyme; the auxiliary enzyme comprises: polyphosphate kinase SlPPK2.
10. Use of the mutant according to any one of claims 2 to 4, the nucleic acid molecule according to claim 5 or 6, the expression vector according to claim 7, the host according to claim 8 and / or the enzyme composition according to claim 9 in the preparation of Arg-Arg dipeptide.
11. A method for preparing an Arg-Arg dipeptide, characterized in that: Mixing the raw material with the mutant according to any one of claims 2 to 4 or the enzyme composition according to claim 9 to obtain Arg-Arg dipeptide; The raw materials include: arginine, magnesium chloride, sodium hexametaphosphate and disodium adenosine triphosphate.
12. The preparation method according to claim 11, characterized in that: The pH value of the mixture is 7.0-9.
5.
13. The preparation method according to claim 11 or 12, characterized in that: The mixing temperature is 25-45°C.
14. The preparation method according to any one of claims 11 to 13, characterized in that: The concentration of the magnesium chloride is 20-80 mM.
15. The preparation method according to any one of claims 11 to 14, characterized in that: The enzyme activity of the mutant is 173 U / mL; the enzyme activity of the auxiliary enzyme is 360 U / mL.