Enzyme mutant and its application in preparing isoleucylarginine

Abstract

The present invention relates to the field of biotechnology, and more particularly to enzyme mutants and their applications in the preparation of isoleucyl arginine. The present invention has discovered an amino acid ligase (Uniprot: A0A2W4HG92) capable of connecting L-isoleucine and L-arginine to synthesize isoleucyl arginine. Based on the enzyme, modification was carried out, and by mutating the amino acid residues in its catalytic activity pocket and other related positions, an enzyme mutant with high catalytic activity was finally obtained, which greatly increased the yield of isoleucyl arginine. This method is significantly superior to chemical synthesis preparation processes, not only increasing the yield of the product, but also showing outstanding advantages in production cost, energy consumption, product quality and green index, and is suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to an enzyme mutant and application thereof in the preparation of isoleucylarginine. Background Art

[0002] Isoleucylarginine is a dipeptide formed by isoleucine (Ile) and arginine (Arg) connected by a peptide bond. It is the active ingredient in fish peptides. It can relieve temporary fatigue in the human body and has cardiovascular support, anti-inflammatory, skin health improvement, antibacterial and antioxidant properties.

[0003] At present, the production process of isoleucyl arginine is mainly based on biological extraction and traditional chemical synthesis. Among them, the natural extraction method is to remove fat and polysaccharides by acid treatment of oyster meat raw materials, denature the protein by alkali treatment, and implement enzymatic hydrolysis and purification treatment using neutral protease and papain. This route has many steps, the process is relatively complicated, and the purification process is relatively difficult. It is greatly affected by natural factors and the overall yield is low (Cai Muyi et al. CN112741199A). The chemical synthesis method includes: first protecting the isoleucine amino group with phthalic anhydride, then activating the carboxyl group with thionyl chloride, and finally condensing the arginine protected by trimethylchlorosilane to obtain protected isoleucyl arginine, and finally deprotecting it. The route is long, the overall yield is low, and it is easy to racemize during the process.

[0004] The aforementioned biological extraction and traditional chemical synthesis methods are currently the primary methods for producing isoleucyl arginine. However, these two methods are characterized by long preparation routes, relatively complex processes, high costs, and low final yields. Both methods also present challenges during industrial scale-up. Therefore, exploring more methods for producing isoleucyl arginine offers significant competitive advantages and practical application value.

[0005] Compared with traditional natural extraction and chemical synthesis processes, biosynthesis offers advantages in terms of ingredient and raw material safety and stability, product bioactivity, and environmental friendliness. However, there are currently no reports of biosynthesizing isoleucyl arginine. Therefore, developing a biosynthetic method for isoleucyl arginine offers significant competitive advantages and practical application value, and will facilitate the large-scale production of isoleucyl arginine. Summary of the Invention

[0006] In view of this, the present invention provides an enzyme mutant and its use in preparing isoleucylarginine.

[0007] The present invention provides an L-amino acid ligase mutant, which has any one single point mutation or a combination of two or more mutations among F9H, H79D, F81D, A153F, K282D and F322R in the amino acid sequence shown in SEQ ID NO: 1.

[0008] This method, using site-directed mutagenesis of the "ATP-grasp" protein (Uniprot: A0A2W4HG92) from Paenibacillus, ultimately achieved the efficient synthesis of an isoleucylarginine dipeptide, which can be used for industrial catalytic production. This method offers numerous advantages, including ease of preparation, high yield, high product quality, good environmental compatibility, low carbon emissions, and ease of scalable production.

[0009] The L-amino acid ligase mutants of the present invention have an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 10, and are named mutants A0A2W4HG92-M01 to A0A2W4HG92-M09 in sequence in the present invention.

[0010] Studies have shown that compared to wild-type L-amino acid ligase, the mutants of the present invention have superior catalytic activity for isoleucyl-arginine synthesis, significantly increasing isoleucyl-arginine production. Among the nine mutants provided herein, A0A2W4HG92-M05, A0A2W4HG92-M07, A0A2W4HG92-M08, and A0A2W4HG92-M09 exhibit the highest and most effective catalytic activity for isoleucyl-arginine synthesis.

[0011] The present invention also provides a biomaterial, characterized in that it comprises at least one of the following:

[0012] 1) a nucleic acid encoding the L-amino acid ligase mutant;

[0013] 2) an expression cassette comprising the nucleic acid described in 1);

[0014] 3) a recombinant vector comprising the nucleic acid described in 1) or the expression cassette described in 2);

[0015] 4) transfecting or transforming a host containing the recombinant vector 3) or a host having 1) the nucleic acid or 2) the expression cassette integrated into its genome.

[0016] In the present invention, nucleic acids encoding L-amino acid ligase mutants as shown in SEQ ID NOs: 2 to 10 are codon-optimized according to the codon preference of the host. In some specific embodiments, the present invention performs codon optimization according to the codon preference of Escherichia coli, and the optimized nucleic acid sequence has any one of the following sequences a) to c):

[0017] a) the nucleotide sequence shown in any one of SEQ ID NOs: 11 to 19; or

[0018] b) a nucleotide sequence obtained by deleting, substituting or adding one or more bases in the nucleotide sequence shown in a), and having the same or similar functions as a); or

[0019] c) A nucleotide sequence having at least 80% homology to the sequence shown in a) or b).

[0020] In the present invention, the host is Escherichia coli.

[0021] The present invention also provides a method for preparing the L-amino acid ligase mutant, which comprises fermenting the host in the biological material to obtain a product containing the L-amino acid ligase mutant.

[0022] The present invention also provides a system for preparing isoleucyl arginine, comprising an ATP regeneration system, and the L-amino acid ligase mutant or the biomaterial.

[0023] The present invention also provides the use of any of the following in the preparation of isoleucyl arginine:

[0024] A. an L-amino acid ligase having the amino acid sequence shown in SEQ ID NO: 1;

[0025] B. the L-amino acid ligase mutant;

[0026] C. the biomaterial of the present invention;

[0027] D. The system for preparing isoleucyl arginine of the present invention.

[0028] In some embodiments, the ATP regeneration system includes polyphosphate kinase (PPK) and hexaphosphate.

[0029] The present invention provides a method for preparing isoleucyl arginine, wherein L-isoleucine and arginine are reacted under the action of L-amino acid ligase to obtain isoleucyl arginine;

[0030] The L-amino acid ligase is selected from any of the following:

[0031] A. an L-amino acid ligase having the amino acid sequence shown in SEQ ID NO: 1;

[0032] B. the L-amino acid ligase mutant of the present invention;

[0033] C. L-amino acid ligase is prepared based on the biomaterial or the system described in the present invention.

[0034] In the present invention, the raw materials for the reaction also include adenosine triphosphate disodium salt. The adenosine triphosphate (ATP) required during the reaction can be an equivalent amount or a catalytic amount (by using an ATP regeneration system, such as polyphosphate kinase (PPK) and hexaphosphate). In a specific embodiment of the present invention, the amino acid sequence of PPK is shown in SEQ ID NO: 21, and the codon-optimized sequence of its encoding gene is shown in SEQ ID NO: 22.

[0035] In the present invention, the reaction concentration of the L-amino acid ligase or the L-amino acid ligase mutant is 1000-2000 U / L, specifically 1000 U / L, 1500 U / L, or 2000 U / L.

[0036] In some embodiments, the molar ratio of L-isoleucine, arginine and adenosine triphosphate disodium salt is 1:(1.1-2):(1.1-2), specifically 1:1.1:1.1 or 1:1.5:2 or 1:2:1.5, or any other value within the above ratio range.

[0037] In some specific embodiments, the concentration of L-isoleucine is 150 mM.

[0038] In some specific embodiments, the concentration of arginine is 165 mM to 300 mM, specifically 165 mM, 225 mM, 300 mM, or any other value within the above two numerical ranges.

[0039] In some specific embodiments, the concentration of the adenosine triphosphate disodium salt is 165 mM to 300 mM, specifically 165 mM, 225 mM, 300 mM, or any other value within the above two numerical ranges.

[0040] In the present invention, the concentration of the L-amino acid ligase is 1000-2000 U / L, specifically 1000, 1200 U / L, 1500 U / L, 1800 U / L, 2000 U / L, or any other value within the above two numerical ranges.

[0041] In the present invention, the reaction temperature is 25-40°C, specifically 25°C, 30°C, 35°C, 40°C, or any other value within the above two numerical ranges.

[0042] In the present invention, the pH of the reaction is 7.0-9.0, specifically 7.0, 8.0 or 9.0, or any other value within the above two numerical ranges.

[0043] In the present invention, the reaction is carried out in a buffer. Utilizing the enzyme mutant of the present invention, two target amino acids (L-isoleucine and arginine) can be directly linked and converted into isoleucylarginine in one step. In some embodiments, the buffer is a Tris-HCl buffer. In some specific embodiments, the Tris-HCl buffer is a 100mM tris-hydroxymethylaminomethane hydrochloric acid solution (i.e., Tris-HCl buffer) with a pH of 8.0.

[0044] The present invention combines rational site-directed mutagenesis with irrational random mutagenesis to construct and screen amino acid ligase mutants, obtaining mutants with excellent catalytic activity and achieving efficient synthesis of isoleucylarginine. The present invention also provides a method for preparing isoleucylarginine using the mutants of the present invention. This method significantly outperforms chemical synthesis processes, not only increasing product yield but also demonstrating outstanding advantages in production cost, energy consumption, product quality, and green index, making it suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 1 shows the liquid chromatogram of Example 1;

[0046] Figure 2 2 shows the mass spectrum of Example 2;

[0047] Figure 3 The results of Ile-Arg production in a 96-well plate reaction for 2 h for wild-type A0A2W4HG92 and mutant enzymes in Example 2 are shown;

[0048] Figure 4 The SDS-PAGE gel electrophoresis results in Example 3 are shown;

[0049] Figure 5 4.2 in Example 4 is shown;

[0050] Figure 6 4.3 in Example 4 is shown;

[0051] Figure 7 4.4 in Example 4 is shown;

[0052] Figure 8 4 shows the liquid chromatogram of Example 5. DETAILED DESCRIPTION

[0053] The invention provides enzyme mutants and their application in the preparation of isoleucyl arginine. Those skilled in the art can learn from this article content, and suitably improve process parameters and realize. It is particularly important to point out that all similar replacements and changes are obvious to those skilled in the art, and they are all considered to be included in the present invention. Method and application of the present invention have been described by preferred embodiment, and relevant personnel obviously can change or suitably change and combine the method and application of this article without departing from the content of the present invention, spirit and scope, to realize and apply the technology of the present invention.

[0054] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

[0055] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0056] The terms "include," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0057] The term "and / or" includes the meanings of "and," "or," and "all or any other combination of elements linked by the subject term."

[0058] The term "homology" as used herein can be calculated in the following manner: to determine the percent "homology" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0059] The term "nucleic acid" as used herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising mixtures of two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms.

[0060] As used herein, the term "vector" or "plasmid vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0061] The term "host" or "host cell" herein refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the initially transformed cell are included herein.

[0062] The invention discloses an amino acid ligase capable of synthesizing isoleucylarginine, a mutant of the enzyme and application thereof.

[0063] The present invention shows through a large number of amino acid ligase catalytic substrate activity screening results that there is an L-amino acid ligase (Uniprot: A0A2W4HG92) in Paenibacillus bacteria that has the ability to connect L-isoleucine and L-isoleucine. According to the enzyme's binding catalytic ability to L-isoleucine, it is deduced that the enzyme is used to catalyze the reaction of L-isoleucine and L-arginine. However, preliminary screening shows that the activity is weak, with almost no activity, and it cannot be directly applied to industrial production. The application also uses the enzyme as a transformation template, combines and utilizes rational site-directed mutagenesis and irrational random mutagenesis, and performs site-directed mutagenesis and superimposed evolution on key sites F9, H79, F87, A153, K282, and F322. Different mutants are screened by the isoleucyl arginine synthesis reaction, and finally a mutant with excellent catalytic activity for synthesizing isoleucyl arginine is obtained, realizing the efficient synthesis of isoleucyl arginine.

[0064]

[0065] This route utilizes inexpensive, non-protecting amino acids as raw materials, and produces high-yield isoleucylarginine in the presence of an equivalent amount of adenosine triphosphate (ATP) and a corresponding amino acid ligase mutant. To further reduce costs, the introduction of an ATP regeneration system into the reaction system can further reduce ATP usage. Overall, this preparation route is streamlined, offers high yields, high product quality, a high green performance index, and is easily scalable.

[0066] The amino acid sequences of the enzymes and mutants involved in the present invention are shown in Table 1:

[0067] Table 1

[0068]

[0069]

[0070] Note: The amino acids in bold and underlined are mutated amino acids. The DNA sequences of the enzymes involved in the present invention are shown in Table 2:

[0071] Table 2

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] Note: The three bases in bold and underlined are the codons after mutation. The test materials used in the present invention are all common commercial products and can be purchased on the market.

[0078] The present invention will be further described below in conjunction with the embodiments:

[0079] Example 1 Preparation of Isoleucylarginine Using Wild-Type Amino Acid Ligase Crude Enzyme

[0080] 1.1 Construction of the A0A2W4HG92 wild-type enzyme expression strain, bacterial culture, and enzyme preparation

[0081] The wild-type L-amino acid ligase from Paenibacillus was codon-optimized according to the preference of Escherichia coli. The encoding gene sequence, SEQ ID NO: 20, was synthesized completely. Restriction endonuclease sites, NdeI and XhoI, were designed at both ends of the gene and subcloned into the corresponding sites of the pET28a vector to generate the recombinant plasmid pET28a-A0A2W4HG92. The recombinant plasmid pET28a-A0A2W4HG92 was transformed into competent E. coli BL21(DE3) cells, the expression host. Single colonies cultured on plates were cultured in small liquid batches. The strains expressing the correct protein were identified as recombinant E. coli A0A2W4HG92-WT expressing the wild-type enzyme.

[0082] A0A2W4HG92 wild-type enzyme culture and enzyme preparation: A single colony of recombinant Escherichia coli A0A2W4HG92-WT was transferred into 5 ml of LB culture medium containing 50 μM kanamycin (37°C) for culture. When the cells reached the logarithmic phase, they were inoculated into 250 ml of LB culture medium containing the same antibiotic. When they also reached the logarithmic phase, they were transferred into a 5 L culture fermenter for culture and final protein expression. In the 5 L fermenter, when the cell OD 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 collected 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 50 mM Tris-HCl buffer (pH 8.0) on ice. The cells were then disrupted by freeze-thaw to obtain the A0A2W4HG92 wild-type enzyme solution. After high-speed centrifugation to remove the cell walls, the supernatant was run on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to confirm protein expression. LB medium was composed of 1% tryptone, 0.5% yeast extract, 1% NaCl, 1% dipotassium phosphate, 1% dipotassium phosphate, and 5% glycerol.

[0083] 1.2 Preparation of Isoleucyl-Arginine Using Ligase (A0A2W4HG92)

[0084] 19.7 g of L-isoleucine (150 mM), 28.7 g of arginine (165 mM), and 90.9 g of adenosine triphosphate disodium salt (ATP, 165 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 a NaOH aqueous solution. 1500 U of ligase A0A2W4HG92 was added to initiate the reaction. The reaction was stirred gently at 30°C while maintaining the pH of the reaction system at 8.0. After 6 hours of reaction, the reaction was detected by HPLC (see Figure 1 ). The pH of the reaction system was adjusted to 2.0 with HCl aqueous solution to denature the enzyme and precipitate it. The protein solid was removed by centrifugation. After the pH of the reaction solution was adjusted to 7.0, it was directly loaded onto a D201 anion exchange resin purification column to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted, concentrated and crystallized using a reverse osmosis membrane (ethanol: water, 2:1, v:v) to obtain 12.9 grams of white isoleucyl arginine solid (yield 30%). The product was confirmed to be isoleucyl arginine by mass spectrometry. The mass spectrometry results are shown in the figure. Figure 2 .

[0085] Example 2: Preparation of Isoleucylarginine Using Crude Enzyme Solution of Mutant Amino Acid Ligase

[0086] This example includes mutation modification, expression, culture, reaction, and comparison of reaction conversion rates of different mutants. The specific process is as follows:

[0087] 2.1 Construction of mutations

[0088] The mutants shown in Table 3 were constructed: A0A2W4HG92-M01, A0A2W4HG92-M02, A0A2W4HG92-M03, A0A2W4HG92-M04, A0A2W4HG92-M05, A0A2W4HG92-M06, A0A2W4HG92-M07, A0A2W4HG92-M08, A0A2W4HG92-M09.

[0089] 2.1.1 Mutation Points and Mutation Schemes

[0090] Table 3

[0091]

[0092]

[0093] 2.1.2 Primer design

[0094] Table 4

[0095]

[0096] 2.1.3 Segmented PCR:

[0097] Using primers with mutations at different sites, segmented PCR is performed to construct gene fragments containing the target mutation. Taking M01, M08, and M09 as an example:

[0098] Table 5

[0099]

[0100]

[0101] PCR reaction system:

[0102] Table 6

[0103] primer PF (10uM) 1ul primer PR (10uM) 1ul Template(LAL16) 0.5ul (20ng / ul) 5X Q5 reaction buffer 5ul 10mM dNTP 0.5ul Q5 polymerase 0.3ul ddH2O Up to 25ul

[0104] PCR machine system:

[0105] Table 7

[0106]

[0107] The PCR amplified products were run on gel electrophoresis, and the PCR-positive bands were recovered and purified.

[0108] 2.1.4 Homologous recombination:

[0109] Recombining and fusing the mutant gene fragments:

[0110] Table 8

[0111] PCR fragment F1 (100 ng / ul) 1 μl 1 μl PCR fragment F2 (100 ng / ul) 1 μl 1 μl PCR fragment F3 (100 ng / ul) / 1 μl 2x seamless cloning Mix 2 μl 3 μl

[0112] Reaction conditions: 50°C for 30 minutes

[0113] 2.1.5 Conversion:

[0114] Take the homologous recombination product and transfer it into 40ul BL21(DE3) competent cells. After ice bath for 30min, heat shock at 42℃ for 45s, ice bath for 2-3min, add 500ul SOC, culture on a shaking platform for 1.5 hours, centrifuge the entire plate, and culture in a 37℃ incubator overnight for 12-16 hours.

[0115] 2.2 96-well plate reaction screening

[0116] 2.2.1 Well plate culture

[0117] To each well of a sterilized 96-well plate, add 400 μL of LB medium containing 50 mg / L Kan. The wild-type A0A2W4HG92 strain was used as a control. Six single colonies from the control and each mutant were selected and incubated in LB medium. Incubate at 37°C, 220 rpm for 5 hours. Add 600 μL of HB medium containing 50 mg / L Kan to each well of the 96-well plate. Transfer 10 μL of activated bacterial suspension from each 96-well plate to HB medium and autoinduce at 37°C, 220 rpm for 24 hours. Measure the OD600, centrifuge at 4000 rpm for 10 minutes, discard the supernatant, and use the remaining pellet in the reaction.

[0118] 2.2.2 Cell disruption

[0119] Prepare 20 mL of lysozyme solution according to the following table:

[0120] Table 9

[0121]

[0122]

[0123] The cultured 96-well plate containing only bacteria was taken out from the -20°C refrigerator, 100 μL of lysozyme solution was added to each well, and the suspended bacteria were thoroughly blown and then broken. The breaking conditions were 37°C, 800 rpm, and 2 h.

[0124] 2.2.3 Reaction and screening

[0125] Prepare 20 mL of 2x reaction solution according to the following table:

[0126] Table 10

[0127] Reagents Reaction concentration / mM Stock solution concentration / mM Theoretical amount / g L-Ile 50 100 0.2623 L-Arg 50 100 0.3484 ATP·2Na 50 100 1.1023 <![CDATA[MgCl2]]> 50 100 0.1906 Tris 50 100 0.2423 <![CDATA[H2O]]> / / Up to 20mL

[0128] Weigh the above reagents and completely dissolve them in 15 mL of water. Add 4 M NaOH to adjust the pH to 9.0, and then adjust the volume to 20 mL. Add 200 μL of 2× reaction solution + 100 μL of H2O to each well, mix well by pipetting, and place on a shaker at 38°C, 800 rpm for 2 hours. Pipette 100 μL of supernatant into each well of a 96-well plate, add 900 μL of 60% acetonitrile to quench the reaction according to a 10-fold dilution, shake at 37°C, 1000 rpm for 10 minutes, and centrifuge at 4000 rpm for 10 minutes. Take the supernatant and filter it with a 0.45 μm filter head for liquid phase detection. The yield of each mutant is shown in the table. Figure 3 .

[0129] 2.3 Reaction results

[0130] according to Figure 3 The yield results of the liquid phase reaction showed that the mutant with the best reaction was the M08 mutant.

[0131] Example 3: Fermentation production of enzymes

[0132] The enzymes required by the present invention are all produced by fermentation in E. coli; the process specifically includes the following steps: plasmid transfer into E. coli (BL21) competent cells for plate culture and single clone screening, small-scale liquid culture of single clones, and finally, step-by-step liquid culture of bacteria with correct protein expression. Specifically, the process includes transferring a single colony 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 5L fermentor for culture and final protein expression. In the 5L fermentor, when the cell OD reaches ~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 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, and then the cells were broken by freeze-thaw method, and the cell walls were removed by high-speed centrifugation. The supernatant was run on SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to determine protein expression (see Figure 4 LB medium consists of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate, and 5% glycerol.

[0133] Example 4: Preparation of Isoleucylarginine by Enzymatic Reaction Using a Ligase Mutant (A0A2W4HG92-M08)

[0134] 4.1 Bacterial cells expressing the correct protein are used for the next catalytic experiment

[0135] The remaining cells were mixed evenly with Tris.HCl buffer (50 mM, pH 8.0) at low temperature (~10 g wet cells: 200 ml buffer mixture), and then the cell walls were broken by low temperature and high pressure. After removing the cell walls by high-speed centrifugation (16000 rpm, 45 min), an enzyme-containing supernatant was obtained for use (the obtained enzyme activity was 500-1000 U / ml, where U is the amount of enzyme required to convert 1 μmol of substrate in one minute at room temperature).

[0136] 4.2 To 3 L of 100 mM Tris-HCl (pH 8.0) solution was added 59.1 g of L-isoleucine (150 mM), 86.2 g of arginine (165 mM), and 272.8 g of adenosine triphosphate disodium salt (ATP, 165 mM). The pH of the reaction system was then adjusted to 8.0-8.5 with aqueous NaOH. Ligase A0A2W4HG92-M08 crude enzyme solution (4000 U) was added to initiate the reaction. The reaction was stirred gently at 30°C while maintaining the pH at 8.0. After 4 hours, the reaction was monitored by HPLC. Figure 5 The pH was then adjusted to 2.0 with aqueous HCl to denature and precipitate the enzyme in the reaction system, and the protein solids were removed by centrifugation. The reaction solution was then adjusted to a pH of 7.0 and directly loaded onto a D201 anion exchange resin purification column to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 2:1, v:v) to obtain 119.0 g of isoleucylarginine solid (yield 92%).

[0137] 4.3 To 3 L of 100 mM Tris-HCl (pH 8.0) solution was added 59.1 g of L-isoleucine (150 mM), 117.6 g of arginine (225 mM), and 496.1 g of adenosine triphosphate disodium salt (ATP, 300 mM). The pH of the reaction system was then adjusted to 8.0-8.5 with aqueous NaOH. Ligase A0A2W4HG92-M08 crude enzyme solution (3000 U) was added to initiate the reaction. The reaction was stirred gently at 40°C while maintaining the pH at 7.0. After 4 hours, the reaction was monitored by HPLC. Figure 6 The pH was then adjusted to 2.0 with aqueous HCl to denature and precipitate the enzyme in the reaction system, and the protein solids were removed by centrifugation. The reaction solution was then adjusted to a pH of 7.0 and directly loaded onto a D201 anion exchange resin purification column to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 2:1, v:v) to yield 121.6 g of isoleucyl arginine solid (94% yield).

[0138] 4.4 To 3 L of 100 mM Tris-HCl (pH 8.0) solution was added 59.1 g of L-isoleucine (150 mM), 156.8 g of arginine (300 mM), and 372.0 g of adenosine triphosphate disodium salt (ATP, 225 mM). The pH of the reaction system was then adjusted to 8.0-8.5 with aqueous NaOH. Ligase A0A2W4HG92-M08 crude enzyme solution (6000 U) was added to initiate the reaction. The reaction was gently stirred at 25°C while maintaining the pH at 8.5. After 4 hours, the reaction was monitored by HPLC. Figure 7 The pH was then adjusted to 2.0 with aqueous HCl to denature and precipitate the enzyme in the reaction system, and the protein solids were removed by centrifugation. The reaction solution was then adjusted to a pH of 7.0 and directly loaded onto a D201 anion exchange resin purification column to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 2:1, v:v) to yield 113.8 g of isoleucylarginine solid (yield 88%).

[0139] Example 5: Preparation of Isoleucyl Arginine Using Ligase (A0A2W4HG92-M08) and ATP Regeneration System

[0140]

[0141] To 3 L of 100 mM Tris-HCl (pH 8.0) solution, add 59.1 g of L-isoleucine (150 mM), 86.1 g of L-arginine (165 mM), 8.3 g of adenosine triphosphate disodium salt (ATP, 5 mM), and 100.9 g of sodium hexaphosphate (55 mM). After adjusting the pH of the solution to 7.5, add 3000 U of polyphosphate kinase PPK enzyme and 4000 U of crude enzyme solution of ligase A0A2W4HG92-M08. Stir gently at 30°C and maintain the pH of the reaction system between 8.0. After 3 hours, the histidine reaction is basically complete as detected by HPLC. The pH of the reaction solution was then adjusted to 2.0 with aqueous HCl to denature and precipitate the enzyme in the reaction system, and the protein solids were removed by centrifugation. The pH of the reaction solution was then adjusted to 7.0 and directly loaded onto a D201 anion exchange resin purification column to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol: water, 2:1, v:v) to obtain 111.07 g of isoleucyl arginine (yield 86%). The test results are shown in FIG. Figure 8 .

[0142] The above are only preferred embodiments 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 the scope of protection of the present invention.

Claims

1. An L-amino acid ligase mutant, wherein in the amino acid sequence shown in SEQ ID NO: 1, only one single point mutation among F9H, H79D, F81D, A153F, K282D, and F322R exists, or any combination mutation shown in (1) to (3) below exists: (1) H79D and F81D; (2) H79D, F81D and F322R; (3) H79D, F81D, A153F ​​and F322R.

2. The L-amino acid ligase mutant according to claim 1, wherein the amino acid sequence is shown in any one of SEQ ID NOs: 2 to 10.

3. Biomaterial, characterized in that Including at least one of 1) to 4): 1) A nucleic acid encoding the L-amino acid ligase mutant according to claim 1 or 2; 2) an expression cassette comprising the nucleic acid described in 1); 3) a recombinant vector comprising the nucleic acid described in 1) or the expression cassette described in 2); 4) transfecting or transforming a host containing 3) the recombinant vector, or a host having 1) the nucleic acid or 2) the expression cassette integrated into its genome.

4. The biomaterial according to claim 3, characterized in that The sequence of the nucleic acid encoding the mutant according to claim 1 or 2 is shown in any one of SEQ ID NOs: 11 to 19.

5. A method for preparing the L-amino acid ligase mutant according to claim 1 or 2, comprising fermenting the host in the biomaterial according to claim 3 or 4 to obtain a product containing the L-amino acid ligase mutant.

6. A system for preparing isoleucyl arginine, characterized in that: Includes an ATP regeneration system and any of the following: The L-amino acid ligase mutant according to any one of claims 1 to 2; The biomaterial according to claim 3 or 4.

7. Use of any of the following in the preparation of isoleucyl arginine: A. The L-amino acid ligase mutant according to any one of claims 1 to 2; B. The biomaterial according to claim 3 or 4; C. The system of claim 6.

8. A method for preparing isoleucyl arginine, characterized in that, Under the action of L-amino acid ligase, L-isoleucine and arginine react to obtain isoleucylarginine; The L-amino acid ligase is selected from any of the following: A. The L-amino acid ligase mutant according to any one of claims 1 to 2; B. L-amino acid ligase is prepared based on the biomaterial according to any one of claims 3 to 4 or the system according to claim 6.

9. The method according to claim 8, characterized in that The raw materials of the reaction also include adenosine triphosphate disodium salt.

10. The method according to any one of claims 8 to 9, characterized in that The concentration of the L-amino acid ligase is 1000-2000 U / L; and / or, The reaction concentration of isoleucine is 150 mM; and / or, The molar ratio of L-isoleucine, arginine and adenosine triphosphate disodium salt is 1: (1.1-2): (1.1-2) and / or, The reaction temperature is 25-40° C., and the pH is 7.0-9.0; and / or, The reaction was carried out in Tris-HCl buffer.

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