Enzyme mutant and application thereof in preparation of isoleucyl arginine

Abstract

The invention relates to the technical field of biology, in particular to an enzyme mutant and application thereof in preparation of isoleucyl arginine. According to the present invention, the amino acid ligase (Uniprot: A0A2W4HG92) capable of connecting the L-isoleucine with the L-arginine so as to synthesize the isoleucyl arginine is found; according to the isoleucyl arginine enzyme, modification is carried out on the basis of the isoleucyl arginine enzyme, a catalytic activity pocket of the isoleucyl arginine enzyme and amino acid residues of other related parts are mutated, an enzyme mutant with high catalytic activity is finally obtained, and the yield of the isoleucyl arginine is greatly increased. The method is obviously superior to a chemical synthesis preparation process, not only improves the yield of the product, but also shows outstanding advantages in the aspects of production cost, energy consumption, product quality and green index, and is suitable for large-scale production.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an enzyme mutant and application thereof in preparing isoleucyl arginine. 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 use neutral protease and papain to implement enzymatic hydrolysis and purification. This route has many steps, the process is relatively complicated, and the purification process is relatively difficult, 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 the 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 above-mentioned biological extraction method and traditional chemical synthesis method are the main methods for producing isoleucyl arginine at present, but these two methods have long preparation routes, relatively complex processes, high costs, low final yields, and some problems in the industrial scale-up process. Therefore, exploring more ways to prepare isoleucyl arginine has great competitive advantages and practical application value.

[0005] Compared with traditional natural extraction and chemical synthesis processes, biosynthesis technology has advantages in terms of safety and stability of ingredients and raw materials, biological activity of products, and environmental friendliness. However, there are no reports on the biological synthesis of isoleucyl arginine. Therefore, the development of a biosynthetic method for isoleucyl arginine has great competitive advantages and practical application value, which is conducive to promoting the large-scale production of isoleucyl arginine. Summary of the invention

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

[0007] The present invention provides an L-amino acid ligase mutant, which has any 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] The present invention performs site-directed mutagenesis on the "ATP-grasp" protein (Uniprot: A0A2W4HG92) from Paenibacillus, and finally realizes the efficient synthesis of isoleucylarginine dipeptide, which can be used for industrial catalytic preparation. The method has many advantages, such as simple preparation, high yield, good product quality, good environmental compatibility, low carbon emissions, and easy large-scale production.

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

[0010] Studies have shown that compared with the wild-type L-amino acid ligase, the mutant of the present invention has excellent catalytic activity of isoleucyl arginine and significantly improves the yield of isoleucyl arginine. Among the 9 mutants provided by the present invention, A0A2W4HG92-M05, A0A2W4HG92-M07, A0A2W4HG92-M08, and A0A2W4HG92-M09 have the highest yield and the best effect in catalyzing the synthesis of isoleucyl arginine.

[0011] The present invention also provides a biomaterial, characterized in that it comprises at least one of 1) to 4):

[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 the nucleic acid 1) or the expression cassette 2) integrated into its genome.

[0016] In the present invention, the nucleic acid encoding the L-amino acid ligase mutants shown in SEQ ID NOs: 2 to 10 is codon optimized according to the host codon preference. 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) a nucleotide sequence as 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, comprising 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 items in the preparation of isoleucyl arginine:

[0024] A. an L-amino acid ligase having an amino acid sequence as 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 an amino acid sequence as 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 of the reaction also include adenosine triphosphate disodium salt. The adenosine triphosphate (ATP) required in the reaction process can be an equivalent amount or a catalytic amount (by matching the ATP regeneration system, using polyphosphate kinase PPK and hexaphosphate). In a specific embodiment of the present invention, the amino acid of the PPK is shown in SEQ ID NO: 21, and the sequence of its coding gene after codon optimization 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. The enzyme mutant of the present invention can be used to directly connect and convert two target amino acids (L-isoleucine and arginine) into isoleucyl arginine 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 mutation and irrational random mutation to construct and screen the amino acid ligase mutant, obtains a mutant with excellent catalytic activity, and realizes efficient synthesis of isoleucyl arginine. The present invention also provides a method for preparing isoleucyl arginine using the mutant of the present invention, which is significantly better than the chemical synthesis preparation process, not only improves the yield of the product, but also shows outstanding advantages in production cost, energy consumption, product quality and green index, and is 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 Ile-Arg production results of wild-type A0A2W4HG92 and mutant enzymes in Example 2 after 2 h of 96-well plate reaction;

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

[0049] Figure 5 The liquid chromatogram of 4.2 in Example 4 is shown;

[0050] Figure 6 The liquid chromatogram of 4.3 in Example 4 is shown;

[0051] Figure 7 The liquid chromatogram of 4.4 in Example 4 is shown;

[0052] Figure 8 The liquid chromatogram of Example 5 is shown. DETAILED DESCRIPTION

[0053] The invention provides enzyme mutants and their application in preparing isoleucyl arginine. Those skilled in the art can learn from this article content and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and the relevant personnel can obviously change or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention, to achieve 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 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 the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that the use of "include", "comprising" and "having" descriptions herein 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 corresponding term".

[0058] The term "homology" herein can be calculated in the following manner: to determine the percentage of "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 comparison 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, the molecules are identical at this position.

[0059] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed 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 a mixture 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] The term "vector" or "plasmid vector" herein 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 operably 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 is introduced, including the offspring of such a cell. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant offspring having the same function or biological activity as screened or selected in the initially transformed cell are included herein.

[0062] The invention discloses an amino acid ligase capable of synthesizing isoleucylarginine, and discloses 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 the bacterium of Paenibacillus (Paenibacillus) with the ability to connect L-isoleucine and L-isoleucine. According to the enzyme's ability to combine and catalyze 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, 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 mutation and irrational random mutation, and performs site-directed mutation and superimposed evolution on key sites F9, H79, F87, A153, K282, and F322, and screens different mutants through isoleucyl arginine synthesis reaction, and finally obtains a mutant with excellent catalytic activity for synthesizing isoleucyl arginine, and realizes the efficient synthesis of isoleucyl arginine.

[0064]

[0065] This route uses cheap amino acids that do not require protective groups as raw materials, and synthesizes isoleucyl arginine with high yield under the action of equivalent adenosine triphosphate (ATP) and corresponding amino acid ligase mutants. In order to further save costs, the introduction of an ATP recycling system in the reaction system can further reduce the amount of ATP used. Therefore, in general, the preparation route is simple, has a high yield, good product quality, a high green index in production, and is easy to scale up.

[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 mutant amino acids. The DNA sequence of the enzyme involved in the present invention is shown in Table 2:

[0071] Table 2

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] Note: The three black and underlined bases are the mutated codons. 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 embodiments:

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

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

[0081] For the wild-type L-amino acid ligase from Paenibacillus, codon optimization was performed according to the preference of Escherichia coli, the coding gene sequence SEQ ID NO: 20 was synthesized, and restriction endonuclease sites NdeI and XhoI were designed at both ends of the gene, and subcloned into the corresponding sites of the vector pET28a to obtain the recombinant plasmid pET28a-A0A2W4HG92. The recombinant plasmid pET28a-A0A2W4HG92 was transformed into the expression host Escherichia coli BL21 (DE3) competent cells, and a small amount of liquid culture was performed on the monoclonal clones cultured on the plate, and the bacteria with correct protein expression were the recombinant Escherichia coli A0A2W4HG92-WT expressing the wild enzyme.

[0082] Bacterial culture and enzyme preparation of A0A2W4HG92 wild enzyme: 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 grew to the logarithmic phase, they were inoculated into 250 ml of LB culture medium containing the same antibiotics. When they also grew to the logarithmic phase, they were transferred into a 5L culture fermenter for culture and final protein expression. In the 5L fermenter culture, when the cell OD was ~ 20, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added at 25°C to induce protein expression for 6 hours, and finally the cells were collected by high-speed centrifugation (4000 rpm, 20 min) to obtain 25-50 g of enzyme overexpression wet cells. Take a small amount of cells and mix them evenly with Tris.HCl buffer (50mM, pH8.0) on an ice basin, then use the freeze-thaw method to break the cells to obtain the A0A2W4HG92 wild enzyme solution, centrifuge at high speed to remove the cell wall, and run SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to determine protein expression. LB medium consists of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen 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), and then the pH value of the reaction system was adjusted to 8.0 with an aqueous NaOH solution, and 1500 U of ligase A0A2W4HG92 was added to start the reaction. The reaction system was stirred slightly at 30°C and the pH of the reaction system was maintained between 8.0. After 6 hours of reaction, HPLC liquid phase detection was performed (see Figure 1 ). The pH was adjusted to 2.0 with HCl aqueous solution to denature the enzyme in the reaction system and precipitate it, and 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. See the mass spectrometry results in the figure Figure 2 .

[0085] Example 2: Preparation of isoleucyl arginine 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 point and mutation scheme

[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 target mutations. Take M01, M08, and M09 as examples:

[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 amplification 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 each mutant gene fragment:

[0110] Table 8

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

[0112] Reaction conditions: 50℃ for 30min

[0113] 2.1.5 Conversion:

[0114] Take the homologous recombination product and transfer it into 40ul BL21(DE3) competent cells. After ice bathing for 30min, heat shock at 42℃ for 45s, ice bath for 2-3min, add 500ul SOC, culture on a shaking table for 1.5 hours, centrifuge the whole 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] Add 400 μL of LB medium containing 50 mg / L Kan to each well of the sterilized 96-well plate. The wild-type A0A2W4HG92 was used as a control. Six single clones of the control and each mutant were picked and placed in LB medium. Culture 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 solution from the 96-well plate to HB medium, and culture for 24 hours at 37°C, 220 rpm for self-induction. Determine OD600, centrifuge at 4000 rpm for 10 minutes, discard the supernatant, and use the remaining precipitated bacteria in the well plate for 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] Take out the cultured 96-well plate containing only bacteria from the -20°C refrigerator, add 100 μL of lysozyme solution to each well, and then blow the suspended bacteria thoroughly to break them. The breaking conditions are 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 in a 96-well plate, add 900 μL of 60% acetonitrile to quench the reaction according to a 10-fold dilution, oscillate 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 um filter head for liquid phase detection. The yield of each mutant is shown in Figure 3 .

[0129] 2.3 Reaction results

[0130] according to Figure 3 From the yield results of the liquid phase reaction, it can be determined that the mutant with the best reaction is the M08 mutant.

[0131] Example 3: Fermentation production of enzymes

[0132] The enzymes required by the present invention are all produced by Escherichia coli fermentation; it specifically includes the following steps: plasmids are transferred into E.coli (BL21) competent cells for plate culture and monoclonal screening, as well as monoclonal small-scale liquid culture, and bacteria with correct protein expression are finally amplified in liquid culture step by step. It specifically includes a single colony transferred into 5ml LB culture solution (37°C) containing 50μM kanamycin for culture, and when the cells grow to the logarithmic phase, they are inoculated into 250ml LB culture solution containing the same antibiotics, and when they grow to the logarithmic phase, they are transferred into a 5L culture fermentation tank for culture and final protein expression. In the 5L fermentation tank culture, when the cell OD is ~ 20, 0.5mM isopropyl-β-D-thiogalactopyranoside (IPTG) is added at 25°C to induce protein expression for 6 hours, and finally the cells are collected by high-speed centrifugation (4000rpm, 20min) to obtain 25-50g of enzyme overexpression wet cells. A small amount of cells was first mixed with Tris-HCl buffer (50 mM, pH 8.0) on an ice basin, and then the cells were broken by freeze-thaw method, and the cell wall was 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% potassium dihydrogen phosphate, 1% potassium dihydrogen phosphate and 5% glycerol.

[0133] Example 4: Preparation of isoleucyl arginine by enzyme-catalyzed reaction using a ligase mutant (A0A2W4HG92-M08)

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

[0135] The remaining cells were mixed evenly with Tris.HCl buffer (50mM, pH8.0) at low temperature (~10g wet cells: 200ml buffer mixture), and then the cell walls were broken by low temperature and high pressure, and the cell walls were removed by high-speed centrifugation (16000rpm, 45min) to obtain the enzyme-containing clear solution for use (the obtained enzyme activity was 500-1000U / ml, U was the amount of enzyme required to convert 1μmol of substrate in one minute at room temperature).

[0136] 4.2 Add 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) to 3 L of 100 mM Tris.HCl solution (pH 8.0), then adjust the pH value of the reaction system to 8.0-8.5 with an aqueous NaOH solution, add ligase A0A2W4HG92-M08 crude enzyme solution (4000 U) to start the reaction, stir lightly at 30°C and maintain the pH of the reaction system between 8.0, and detect the reaction by HPLC after 4 hours. Figure 5 Then, the pH was adjusted to 2.0 with HCl aqueous solution to denature the enzyme in the reaction system and precipitate it, and 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 119.0 g of isoleucyl arginine solid (yield 92%).

[0137] 4.3 Add 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) to 3 L of 100 mM Tris-HCl solution (pH 8.0), then adjust the pH value of the reaction system to 8.0-8.5 with an aqueous NaOH solution, add ligase A0A2W4HG92-M08 crude enzyme solution (3000 U) to start the reaction, stir lightly at 40°C and maintain the pH of the reaction system between 7.0, and detect the reaction by HPLC after 4 hours. Figure 6 Then, the pH was adjusted to 2.0 with HCl aqueous solution to denature the enzyme in the reaction system and precipitate it, and 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 121.6 grams of isoleucyl arginine solid (yield 94%).

[0138] 4.4 Add 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) to 3 L of 100 mM Tris-HCl solution (pH 8.0), then adjust the pH value of the reaction system to 8.0-8.5 with an aqueous NaOH solution, add ligase A0A2W4HG92-M08 crude enzyme solution (6000 U) to start the reaction, stir lightly at 25°C and maintain the pH of the reaction system between 8.5, and detect the reaction by HPLC after 4 hours. Figure 7 Then, the pH was adjusted to 2.0 with HCl aqueous solution to denature the enzyme in the reaction system and precipitate it, and 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 113.8 grams of isoleucyl arginine solid (yield 88%).

[0139] Example 5: Preparation of isoleucyl-arginine using ligase (A0A2W4HG92-M08) and ATP regeneration system

[0140]

[0141] In 3L 100mM pH8.0 tris(hydroxymethylaminomethane)hydrochloric acid (Tris.HCl) solution, add 59.1g L-isoleucine (150mM), 86.1g L-arginine (165mM), 8.3g adenosine triphosphate disodium salt (ATP, 5mM) and 100.9g sodium hexaphosphate (55mM), adjust the pH value of the solution to 7.5, add 3000U of polyphosphate kinase PPK enzyme and 4000U of ligase A0A2W4HG92-M08 crude enzyme solution, stir gently at 30℃ and maintain the pH of the reaction system between 8.0. After 3 hours, it was detected by HPLC that the histidine reaction was basically complete. Then, the pH of the reaction solution was adjusted to 2.0 with HCl aqueous solution to denature the enzyme in the reaction system and precipitate it, and 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 111.07 g of isoleucyl arginine (yield 86%). The test results are shown in 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, there is any single point mutation of F9H, H79D, F81D, A153F, K282D, F322R or a combination of two or more mutations. 2 . The L-amino acid ligase mutant according to claim 1 , which has an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 10. 3 .

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 the recombinant vector described in 3) or a host having the nucleic acid described in 1) or the expression cassette described in 2) integrated into its genome.

4. The biomaterial according to claim 3, characterized in that The nucleic acid encoding the mutant according to claim 1 or 2 has: a) a nucleotide sequence as shown in any one of SEQ ID NOs: 11 to 19; or 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 c) A nucleotide sequence having at least 80% homology to the sequence shown in a) or b).

5. A method for preparing the L-amino acid ligase mutant according to claim 1 or 2, comprising fermenting the host in the biological material 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. an L-amino acid ligase having an amino acid sequence as shown in SEQ ID NO: 1; B. The L-amino acid ligase mutant according to any one of claims 1 to 2; C. The biological material according to claim 3 or 4; D. 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. an L-amino acid ligase having an amino acid sequence as shown in SEQ ID NO: 1; B. The L-amino acid ligase mutant according to any one of claims 1 to 2; C. 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 for 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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