High-thermal-stability deoxyadenosine kinase mutant and application thereof

Abstract

The invention provides a high-thermal-stability deoxyadenosine kinase mutant and application thereof, and belongs to the field of enzyme engineering. Compared with an amino acid sequence as shown in SEQ ID NO.1, the high-thermal-stability deoxyadenosine kinase mutant provided by the invention has mutation of at least one amino acid site, and the mutation comprises V40G, D121T and S154L. In the high-thermal-stability deoxyadenosine kinase mutant provided by the invention, the half-life period of V40G at 50 DEG C is 1.02 h, the half-life period of D121T at 50 DEG C is 9.51 h, and the half-life period of S154L at 50 DEG C is 5.23 h, which are significantly higher than those of a wild type. The high-thermal-stability deoxyadenosine kinase provided by the invention can be used for enzymatic preparation of 2 '-deoxyadenosine monophosphate, deoxyadenosine and deoxyadenosine triphosphate react to obtain deoxyadenosine monophosphate by adopting a one-pot enzymatic method, and the high-thermal-stability deoxyadenosine kinase is simple to operate, can tolerate higher reaction temperature and has the advantage of industrial amplification.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme engineering, and particularly relates to a thermostable deoxyadenosine kinase mutant and its application. Background Art

[0002] Deoxyadenylate is an important component of deoxyribonucleic acid and is widely used in fields such as genetic engineering and drug research and development. Analogs obtained by changing the chemical structure of deoxyadenylate have good anti-tumor activity, such as Fludarabine used for the treatment of chronic lymphocytic leukemia and Cladribin used for the treatment of non-Hodgkin lymphoma. In addition, deoxyadenosine is an intermediate in the synthesis of anti-human immunodeficiency virus (HIV) drugs.

[0003] Currently, the preparation of deoxyadenylate mainly includes deoxyribonucleic acid degradation method, chemical synthesis method and biocatalytic method. In the deoxyribonucleic acid degradation method, the substrate deoxyribonucleic acid has poor stability, is prone to produce a large number of by-products during the preparation process, and has a low yield. The chemical synthesis method cannot meet the current market demand due to high preparation cost and environmental unfriendliness.

[0004] In contrast, the biocatalytic method for preparing deoxyadenylate has significant advantages. It can carry out reactions under mild conditions and avoids the use of toxic reagents. However, most of the existing technologies adopt whole-cell synthesis method. This method uses intact living cells as biocatalysts and completes the synthesis of target products through the enzyme system and metabolic pathways in the cells. However, due to the complex cell metabolism, it is necessary to additionally pay attention to the generation of by-products and the removal methods of various impurities.

[0005] The cell-free synthesis method does not rely on intact cells, but uses cell extracts or purified enzyme systems to complete the synthesis in vitro, with fewer by-products and impurities. However, there are still other disadvantages, especially the poor thermal stability of deoxyadenosine kinase. This enzyme is easily inactivated under high-temperature conditions. During the reaction with wild enzyme, after 5 hours of reaction, the conversion rate no longer changes, and it is necessary to add enzyme to continue the reaction. This leads to a decrease in reaction efficiency. Therefore, the research and development of thermostable deoxyadenosine kinase is of great significance for improving production efficiency and reducing costs. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a thermostable deoxyadenosine kinase mutant and its application.

[0007] On the one hand, the present invention provides a thermostable deoxyadenosine kinase mutant.

[0008] Specifically, compared with the amino acid sequence shown in SEQ ID NO.1, the highly thermostable deoxyadenosine kinase mutant includes at least one of the following mutations: V40G, D121T, and S154L.

[0009] SEQ ID NO.1: mttpilnssvpgniqkkslegthiaisgligagkttlavalgkvlnlptyfeevidnlylqdfykdpkkygfqlqiyllnsrfqqqqqiiwqarggvqdrtiyedsvfakmlnesgllddrdyntycklfqnlsnfmrrpdliihldvspeksleriklrnrdcekdvsleylqnlynayheflqdisryipvirinwsefvdpeqlaqmikaeyesmrfmnqinpptfgngpttnkiistpkdl.

[0010] On the other hand, the present invention provides a nucleic acid.

[0011] Specifically, the nucleic acid encodes the above-mentioned highly thermostable deoxyadenosine kinase mutant.

[0012] In yet another aspect, the present invention provides an expression cassette.

[0013] Specifically, the expression cassette includes the above nucleic acid.

[0014] More specifically, the expression cassette contains a promoter, and the promoter is a eukaryotic promoter and / or a prokaryotic promoter. The eukaryotic promoter is selected from one or more of CMV, SV40, CAG, GAL1, GAL10, TEF1, or TRE, and the prokaryotic promoter is selected from one or more of T7, T3, araBAD, lac, and trp.

[0015] In still another aspect, the present invention provides a genetic engineering vector.

[0016] Specifically, the genetic engineering vector is a eukaryotic expression vector or a prokaryotic expression vector. The eukaryotic expression vector is selected from one or more of pFastBac, pEGFP-N1, pcDNA3.1, and pLVX-CMV-MCS-Linker-EGFP-IRES-Puro, and the prokaryotic expression vector is selected from one or more of pET28a, pET32a, pGEX-4T, pMAL-p2x, and pMAL-c2X.

[0017] In certain specific embodiments of the present invention, the genetic engineering vector is pET28a(+).

[0018] On the other hand, the present invention provides a microorganism.

[0019] Specifically, the microorganism contains the above-mentioned thermostable deoxyadenosine kinase mutant, nucleic acid, expression cassette, and / or genetic engineering vector.

[0020] More specifically, the microorganism is Escherichia coli W3110, BL21(DE3), BW25113, or Rosetta.

[0021] Preferably, the microorganism is Escherichia coli BL21(DE3).

[0022] On the other hand, the present invention provides a method for preparing the above-mentioned thermostable deoxyadenosine kinase mutant.

[0023] Specifically, the preparation method includes the following steps: (1) Construct a genetic engineering vector; (2) Perform site-directed mutagenesis of V40G, D121T, and / or S154L by PCR to obtain the coding gene of the thermostable deoxyadenosine kinase mutant; (3) Express and purify the thermostable deoxyadenosine kinase mutant.

[0024] More specifically, in step (2), the primer sequences used for V40G site-directed mutagenesis are as shown in SEQ ID NO.4-5; the primer sequences used for D121T site-directed mutagenesis are as shown in SEQ ID NO.18-19; the primer sequences used for S154L site-directed mutagenesis are as shown in SEQ ID NO.30-31.

[0025] On the other hand, the present invention provides the application of the above-mentioned thermostable deoxyadenosine kinase mutant, nucleic acid, expression cassette, genetic engineering vector, and / or microorganism in the preparation of deoxyadenylic acid.

[0026] On the other hand, the present invention provides a method for preparing deoxyadenylic acid.

[0027] Specifically, the method uses the above-mentioned thermostable deoxyadenosine kinase mutant and / or microorganism as a catalyst.

[0028] More specifically, the method uses deoxyadenosine, deoxyadenosine triphosphate, and magnesium chloride hexahydrate as substrates, and the thermostable deoxyadenosine kinase and adenylate kinase as catalysts to synthesize deoxyadenylic acid in a one-step reaction.

[0029] Preferably, the final concentration of deoxyadenosine is 200 - 400 mM, the final concentration of deoxyadenosine triphosphate is 110 - 240 mM, the final concentration of magnesium chloride hexahydrate is 50 - 80 mM, the addition amount of the thermostable deoxyadenosine kinase is 50,000 - 60,000 U / mL, and the addition amount of adenylate kinase is 18,000 - 25,000 U / mL.

[0030] In certain specific embodiments of the present invention, the one-step reaction is carried out at 37 - 55 °C.

[0031] Compared with the prior art, the present invention has the following advantages: 1. The thermostable deoxyadenosine kinase mutant provided by the present invention can survive for a long time at 50 °C, and the survival time is increased by 3 - 37 times compared with the wild-type deoxyadenosine kinase.

[0032] 2. The thermostable deoxyadenosine kinase mutant provided by the present invention can be used for the one-pot enzymatic reaction of deoxyadenosine and deoxyadenosine triphosphate to deoxyadenosine monophosphate, with simple operation. The reaction system provided by the present invention can carry out the reaction at 50 °C, having the advantage of industrial scale-up. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a three-dimensional simulation diagram of dAK.

[0034] Figure 2 It is the experimental result of the thermostability exploration experiment of deoxyadenosine kinase. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The following combines specific embodiments to further elaborate on the present invention. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions indicated in the embodiments are usually carried out under conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0036] Basic Example 1 Cloning and Expression of Deoxyadenosine Kinase The deoxyadenosine kinase (nucleotide sequence: Dictyostelium discoideum deoxyadenosine kinase (DAK) mRNA, complete cds, GenBank: AY192983.1) was used. The rare codons in these nucleotide sequences were optimized according to the E. coli codon preference. An NcoⅠ restriction site (CCATGG, since the second codon starts with G, only CC was added) was added before the start codon, the stop codon was deleted, and an XhoⅠ restriction site was added at the end of the sequence. The sequence was fully gene synthesized by Shanghai Jierui and ligated to the pET28a(+) vector using the NcoⅠ / XhoⅠ double restriction sites to construct the pET28a(+)-DddAK expression vector. The constructed plasmid was centrifuged at 10,000 rpm for 10 min, and 50 μL of ddH2O was added and dissolved on a vortex shaker. After dissolution, it was transformed into Escherichia coli E. coli BL21(DE3), and the transformed Escherichia coli E. coli BL21(DE3) was activated, placed at 37 ºC and 220 rpm, cultured for 1 hour, spread on an LB plate containing 50 μg / mL kanamycin resistance, and cultured overnight at 37 ºC in an inverted position. A single colony was picked from the plate and inoculated into TB liquid medium containing 50 μg / mL kanamycin at a final concentration, and glucose at a final concentration of 5 g / L and lactose at 20 g / L were added. It was cultured at 37 ºC and 220 rpm until the OD600 reached 0.8 - 1.2 (preferably 0.9), the temperature was changed to 28 ºC, and after culturing at 220 rpm for 14 h, it was centrifuged at 4 ºC and 8000 rpm for 10 minutes to obtain wet bacterial cells containing deoxyadenosine kinase.

[0037] Basic Example 2 Purification of Deoxyadenosine Kinase The wet bacterial cells obtained from Basic Example 1 were suspended in Buffer A (pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 20 mM imidazole), sonicated for 15 minutes (ice bath, power 400 W, sonication for 3 seconds and pause for 5 seconds), centrifuged at 12,000 rpm for 20 min at 4°C, and the supernatant was taken. Ni affinity column (1.6 × 10 cm, Bio-Rad, USA) was used to purify the protein. The specific operations were as follows: ① Equilibrate the Ni column with 5 column volumes of binding buffer (pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 20 mM imidazole) until the baseline was stable; ② Load the sample at a flow rate of 1 mL / min, with the loading amount of 25 - 40 mg / mL protein, so that the target protein was adsorbed onto the Ni column; ③ Wash the impurity proteins with 6 column volumes of Buffer A (pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 20 mM imidazole) at a flow rate of 1 mL / min until the baseline was stable; ④ Elute with Buffer B (pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 400 mM imidazole) at a flow rate of 1 mL / min, and collect the target protein. The target protein was dialyzed overnight in 20 mM Tris buffer at pH 8.0 to obtain purified deoxyadenosine kinase; ⑤ Wash the Ni column with 5 column volumes of binding buffer (pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl) until the baseline was stable, and store the Ni column with 5 column volumes of ultrapure water containing 20% ethanol.

[0038] Basic Example 3 Determination of Enzyme Activity and Thermal Stability of Deoxyadenosine Kinase The definition of enzyme activity unit (U) is: under the conditions of 35 °C and pH 8.0, the amount of enzyme required to generate 1 µmol of dAMP per minute is defined as one enzyme activity unit, U.

[0039] Standard conditions for enzyme activity detection: 5.025 g / L deoxyadenosine, 12.815 g / L ATP-Na2, 16.27 g / L MgCl2∙6H2O, 6.05 g / L Tris, appropriate amount of enzyme solution, react for 15 minutes under the conditions of 30 °C and pH 7.5, and the sample was processed and analyzed by HPLC.

[0040] Liquid phase detection conditions for deoxyadenosine, deoxyadenylic acid, adenosine diphosphate and adenosine triphosphate: Chromatographic column InertSustainTM C18 (4.6 × 150 mm, 5 µm, Shimadzu) column, mobile phase acetonitrile: 0.1 M PBS = 5:95, flow rate 1.0 mL / min, detection wavelength 259 nm, injection volume 10 μL, column temperature 25 °C.

[0041] Investigation on the thermal stability of deoxyadenosine kinase: After diluting deoxyadenosine kinase 100-fold, it was placed at 35 °C, 40 °C, and 50 °C respectively. Samples were taken at different times to measure the enzyme activity. The results are as Figure 1 shown: The enzyme activity of adenosine kinase remained about 55% after overnight incubation at 35 °C, about 11% after overnight incubation at 40 °C, and decreased to about 10% of the original after 1 h at 50 °C, indicating poor thermal stability.

[0042] Example 1 Construction, expression, and screening of the dAK mutant library 1. Molecular dynamics simulation, multiple sequence alignment of dAK, and screening principle of the mutant library (1) Use SWISS-MODEL to predict the three-dimensional structure of DAK. Align the amino acid sequence of DAK with the amino acid sequence of the template protein, and select the three-dimensional model with the highest GMQE score for modeling, as Figure 1 shown.

[0043] (2) Use GROMACS for molecular dynamics (MD) simulation. First, prepare the protein complex file with ligands after YASARA docking. Subsequently, through creating a topology file, defining the simulation box, adding solvents and ions, energy minimization, NPT and NVT equilibration preprocessing, modify the parameters to be simulated in the md.mdp file, perform MD simulation, generate a trajectory file, and finally generate RMSD and RMSF by inputting commands. Through RMSD analysis, the overall structural stability of the protein during the kinetic simulation can be evaluated, while analyzing RMSF helps to identify flexible regions in the protein.

[0044] (3) In the NCBI database, use the protein blast tool with the amino acid sequence of DAK as a probe to query enzyme proteins highly similar to the DAK sequence. Select all amino acid sequences with an identity greater than 40% to the DAK sequence for multiple sequence alignment. Use the ESPript 3.0 tool to perform multiple sequence alignment of these homologous proteins. By aligning a series of homologous protein sequences, amino acid residues that are highly conserved during evolution can be identified. These conserved residues are often closely related to the activity, binding site, or structural stability of the protein. In protein engineering, by purposefully modifying these key residues, the activity, selectivity, or stability of the protein can be changed.

[0045] Combining the methods of molecular dynamics simulation and protein multiple sequence alignment, select important amino acid residue sites to construct a mutant library. The screening principle is as follows: 1) Analyzing the RMSF helps identify flexible regions in the protein structure. These flexible regions include the active site of DAK and relatively active flexible regions. Excluding the relevant active sites and mutating these amino acid residues with large fluctuations into other amino acid residues may help improve the stability of DAK.

[0046] 2) Protein multiple sequence alignment can reveal the evolutionary homology of related proteins. It provides key information for understanding the functional regions, active sites, domains of proteins and their conservation in different species, and is crucial for designing and modifying proteins to improve their activity, stability or change their specific functions. Selecting the relatively conserved amino acid residue sites and mutating them into other amino acid residues with a higher frequency in homologous proteins, or mutating them into other amino acid residues with significant physicochemical property differences from thermophilic microorganisms, is expected to improve the thermal stability of dAK.

[0047] 2. Construction of the dAK mutant library Based on the above site selection principle, the potentially beneficial mutant sites predicted by computer simulation technology are: L30N, V40G, A41M, L42I, L59P, P68M, K70R, L79F, D121T, D123E, Y124F, N125K, T126Q, H146Y, S154L, N162G, V169I, Y178N, H182E.

[0048] The construction method of the above 19 single-point mutants is as follows: Using the pET28a-DddAK plasmid in Basic Example 1 as a template, adopting the principle of Quick-change site-directed mutagenesis, and using the primers listed in Table 1 to mutate the target amino acids to construct a mutant library.

[0049] Table 1 Primer sequence list

[0050] PCR amplification uses Takara's KOD high-fidelity polymerase, and the amplification conditions are: 95°C for 2 min, then 55°C for 20 sec, 72°C for 100 sec, for a total of 30 cycles, and finally 72°C for 10 min. Gel-purify the PCR product, digest the gel-purified product with DpnI enzyme (Fermentas) at 37°C for 2 h to degrade the initial template. Transform the digested product into BL21(DE3), coat it on an LB agar plate containing 50 μg / mL kanamycin, culture it overnight at 37°C, screen positive clones, verify by sequencing, and retain the recombinant strains with correct sequencing results.

[0051] 3. Expression and screening of mutant engineering bacteria The recombinant strain obtained in the previous step was processed according to the method of Basic Example 1 to obtain a crude enzyme solution of the dAK enzyme mutant, and the thermal stability of each mutant at 50 °C was measured according to the method of Basic Example 3. The results are shown in Table 2.

[0052] Table 2

[0053] Among all the mutants, the half-life of V40G at 50 °C was 1.02 h, the half-life of D121T at 50 °C was 9.51 h, and the half-life of S154L at 50 °C was 5.23 h. The crude enzyme solutions of the above mutants were purified according to the method of Basic Example 2 to obtain the corresponding pure enzyme solutions of the mutants for subsequent experiments.

[0054] Experimental Example 1 Application Example of Wild-Type Deoxyadenosine Kinase The wild-type deoxyadenosine kinase was applied to the preparation of deoxyadenylic acid. Using deoxyadenosine and deoxyadenosine triphosphate as raw materials, the reaction system was 100 mL. The addition amount of deoxyadenosine was 5.03 g (200 mM), the addition amount of deoxyadenosine triphosphate was 7.76 g (110 mM), the molar ratio of deoxyadenosine to deoxyadenosine triphosphate was 2:1.1, the addition amount of magnesium chloride hexahydrate was 1.42 g (70 mM), and the pH was 9.0. The addition amount of deoxyadenosine kinase (source: Phorcysia thermohydrogeniphila, The amino acid sequence is mikvvflgppgagkgtqavriaekynvphistgdilraavkegtelgklaksymdrgelvpdeviigiirerlsqpdvkergfildgfprtiaqaealdellkelqlpldkviylnvddeeivkrllargraddtedvirnrlkvyreqtaplvdyytekgllveicgvgeieeitkkieeslglnd, the same below) was 0.56×10⁴ U, and the addition amount of adenylate kinase was 1.21×10⁴ U. The reaction was carried out statically at 50 °C, and the pH was adjusted back to 8.5 with NaOH solution every 1 h of reaction. Samples were taken during the reaction process and analyzed by HPLC according to the liquid phase detection conditions of Basic Example 1. The final conversion rate of the reaction was 37.234%. The conversion rate hardly changed after 8 h of reaction.

[0055] Experimental Example 2 Application Example of Deoxyadenosine Kinase Mutant with High Thermal Stability The highly thermostable deoxyadenosine kinase mutant (V40G) was applied to the preparation of deoxyadenylic acid. Using deoxyadenosine and deoxyadenosine triphosphate as raw materials, the reaction system was 100 mL. The addition amount of deoxyadenosine was 5.03 g (200 mM), the addition amount of deoxyadenosine triphosphate was 7.76 g (110 mM), the molar ratio of deoxyadenosine to deoxyadenosine triphosphate was 2:1.1, the addition amount of magnesium chloride hexahydrate was 1.42 g (70 mM), and the pH was 9.0. The addition amount of deoxyadenosine kinase was 0.56×10⁴ U, and the addition amount of adenylate kinase was 1.21×10⁴ U. The reaction was carried out statically at 50 °C, and the pH was adjusted back to 8.5 with NaOH solution every 1 h of reaction. Samples were taken during the reaction process and analyzed by HPLC according to the liquid phase detection conditions of Basic Example 1. The final conversion rate of the reaction was 81.276%. The final concentration of deoxyadenylic acid was 252 mM.

[0056] Experimental Example 3 Application Example of Highly Thermostable Deoxyadenosine Kinase Mutant The highly thermostable deoxyadenosine kinase mutant (V40G) was applied to the preparation of deoxyadenylic acid. Using deoxyadenosine and deoxyadenosine triphosphate as raw materials, the reaction system was 300 mL. The addition amount of deoxyadenosine was 30.19 g (400 mM), the addition amount of deoxyadenosine triphosphate was 46.55 g (220 mM, and NaOH was continuously added during the dissolution process), the molar ratio of deoxyadenosine to deoxyadenosine triphosphate was 2:1.1, the addition amount of magnesium chloride hexahydrate was 4.27 g (70 mM), and the pH was 9.0. The addition amount of deoxyadenosine kinase was 1.68×10⁴ U, and the addition amount of adenylate kinase was 3.63×10⁴ U. The reaction was carried out statically at 50 °C, and the pH was adjusted back to 8.5 with NaOH solution every 1 h of reaction. Samples were taken during the reaction process and analyzed by HPLC according to the liquid phase detection conditions of Basic Example 1. The final conversion rate of the reaction after 80 h was dAMP 88.452%. The final concentration of deoxyadenylic acid was 548 mM.

[0057] Experimental Example 4 Application Example of Highly Thermostable Deoxyadenosine Kinase Mutant The highly thermostable deoxyadenosine kinase mutant (V40G) was applied to the preparation of deoxyadenylic acid. Using deoxyadenosine and deoxyadenosine triphosphate as raw materials, the reaction system was 300 mL. The addition amount of deoxyadenosine was 30.19 g (400 mM), the addition amount of deoxyadenosine triphosphate was 50.78 g (240 mM, and NaOH was continuously added during the dissolution process). The molar ratio of deoxyadenosine to deoxyadenosine triphosphate was 2:1.2. The addition amount of magnesium chloride hexahydrate was 4.27 g (70 mM), and the pH was 9.0. The addition amount of deoxyadenosine kinase was 16,800 U, and the addition amount of adenylate kinase was 36,300 U. The reaction was carried out statically at 50 °C, and the pH was adjusted back to 8.5 with NaOH solution every 1 h. Samples were taken during the reaction process and analyzed by HPLC according to the liquid phase detection conditions of Basic Example 1. The final conversion rate after 60 h of reaction was 84.890% of dAMP. The final concentration of deoxyadenylic acid was 543 mM.

[0058] Experimental Example 5 Application Example of Highly Thermostable Deoxyadenosine Kinase Mutant The highly thermostable deoxyadenosine kinase mutant (D121T) was applied to the preparation of deoxyadenylic acid. Using deoxyadenosine and deoxyadenosine triphosphate as raw materials, the reaction system was 100 mL. The addition amount of deoxyadenosine was 5.03 g (200 mM), the addition amount of deoxyadenosine triphosphate was 7.76 g (110 mM). The molar ratio of deoxyadenosine to deoxyadenosine triphosphate was 2:1.1. The addition amount of magnesium chloride hexahydrate was 1.42 g (70 mM), and the pH was 9.0. The addition amount of deoxyadenosine kinase was 5,600 U, and the addition amount of adenylate kinase was 12,100 U. The reaction was carried out statically at 50 °C, and the pH was adjusted back to 8.5 with NaOH solution every 1 h. Samples were taken during the reaction process and analyzed by HPLC according to the liquid phase detection conditions of Basic Example 1. The final conversion rate of the reaction was 80.897%. The final concentration of deoxyadenylic acid was 251 mM.

[0059] Experimental Example 6 Application Example of Highly Thermostable Deoxyadenosine Kinase Mutant The highly thermostable deoxyadenosine kinase mutant (D121T) was applied to the preparation of deoxyadenylic acid. Using deoxyadenosine and deoxyadenosine triphosphate as raw materials, the reaction system was 300 mL. The addition amount of deoxyadenosine was 30.19 g (400 mM), the addition amount of deoxyadenosine triphosphate was 46.55 g (220 mM, and NaOH was continuously added during the dissolution process). The molar ratio of deoxyadenosine to deoxyadenosine triphosphate was 2:1.1. The addition amount of magnesium chloride hexahydrate was 4.27 g (70 mM), and the pH was 9.0. The addition amount of deoxyadenosine kinase was 16,800 U, and the addition amount of adenylate kinase was 36,300 U. The reaction was carried out statically at 50 °C, and the pH was adjusted back to 8.5 with NaOH solution every 1 h of reaction. Samples were taken during the reaction process and analyzed by HPLC according to the liquid phase detection conditions of Basic Example 1. The final conversion rate after 80 h of reaction was dAMP 88.934%. The final concentration of deoxyadenylic acid was 551 mM.

[0060] Experimental Example 7 Application Example of Highly Thermostable Deoxyadenosine Kinase Mutant The highly thermostable deoxyadenosine kinase mutant (D121T) was applied to the preparation of deoxyadenylic acid. Using deoxyadenosine and deoxyadenosine triphosphate as raw materials, the reaction system was 300 mL. The addition amount of deoxyadenosine was 30.19 g (400 mM), the addition amount of deoxyadenosine triphosphate was 50.78 g (240 mM, and NaOH was continuously added during the dissolution process). The molar ratio of deoxyadenosine to deoxyadenosine triphosphate was 2:1.2. The addition amount of magnesium chloride hexahydrate was 4.27 g (70 mM), and the pH was 9.0. The addition amount of deoxyadenosine kinase was 16,800 U, and the addition amount of adenylate kinase was 36,300 U. The reaction was carried out statically at 50 °C, and the pH was adjusted back to 8.5 with NaOH solution every 1 h of reaction. Samples were taken during the reaction process and analyzed by HPLC according to the liquid phase detection conditions of Basic Example 1. The final conversion rate after 60 h of reaction was dAMP 85.231%. The final concentration of deoxyadenylic acid was 545 mM.

[0061] Experimental Example 8 Application Example of Highly Thermostable Deoxyadenosine Kinase Mutant The highly thermostable deoxyadenosine kinase mutant (S154L) was applied to the preparation of deoxyadenylic acid. Using deoxyadenosine and deoxyadenosine triphosphate as raw materials, the reaction system was 100 mL. The addition amount of deoxyadenosine was 5.03 g (200 mM), the addition amount of deoxyadenosine triphosphate was 7.76 g (110 mM), the molar ratio of deoxyadenosine to deoxyadenosine triphosphate was 2:1.1, the addition amount of magnesium chloride hexahydrate was 1.42 g (70 mM), and the pH was 9.0. The addition amount of deoxyadenosine kinase was 0.56×10⁴ U, and the addition amount of adenylate kinase was 1.21×10⁴ U. The reaction was carried out statically at 50 °C, and the pH was adjusted back to 8.5 with NaOH solution every 1 h. Samples were taken during the reaction and analyzed by HPLC according to the liquid phase detection conditions of Basic Example 1. The final conversion rate of the reaction was 80.432%. The final concentration of deoxyadenylic acid was 249 mM.

[0062] Experimental Example 9 Application Example of Highly Thermostable Deoxyadenosine Kinase Mutant The highly thermostable deoxyadenosine kinase mutant (S154L) was applied to the preparation of deoxyadenylic acid. Using deoxyadenosine and deoxyadenosine triphosphate as raw materials, the reaction system was 300 mL. The addition amount of deoxyadenosine was 30.19 g (400 mM), the addition amount of deoxyadenosine triphosphate was 46.55 g (220 mM, and NaOH was continuously added during the dissolution process), the molar ratio of deoxyadenosine to deoxyadenosine triphosphate was 2:1.1, the addition amount of magnesium chloride hexahydrate was 4.27 g (70 mM), and the pH was 9.0. The addition amount of deoxyadenosine kinase was 1.68×10⁴ U, and the addition amount of adenylate kinase was 3.63×10⁴ U. The reaction was carried out statically at 50 °C, and the pH was adjusted back to 8.5 with NaOH solution every 1 h. Samples were taken during the reaction and analyzed by HPLC according to the liquid phase detection conditions of Basic Example 1. The final conversion rate of the reaction after 80 h was 87.689% for dAMP. The final concentration of deoxyadenylic acid was 544 mM.

[0063] Experimental Example 10 Application Example of Highly Thermostable Deoxyadenosine Kinase Mutant The thermostable deoxyadenosine kinase mutant (S154L) was applied to the preparation of deoxyadenylic acid. Using deoxyadenosine and deoxyadenosine triphosphate as raw materials, the reaction system was 300 mL. The addition amount of deoxyadenosine was 30.19 g (400 mM), the addition amount of deoxyadenosine triphosphate was 50.78 g (240 mM, and NaOH was continuously added during the dissolution process). The molar ratio of deoxyadenosine to deoxyadenosine triphosphate was 2:1.2. The addition amount of magnesium chloride hexahydrate was 4.27 g (70 mM), and the pH was 9.0. The addition amount of deoxyadenosine kinase was 16,800 U, and the addition amount of adenylate kinase was 36,300 U. The reaction was carried out statically at 50 °C, and the pH was adjusted back to 8.5 with NaOH solution every 1 h. Samples were taken during the reaction process and analyzed by HPLC according to the liquid phase detection conditions of Basic Example 1. The final conversion rate after 60 h of reaction was 84.530% of dAMP. The final concentration of deoxyadenylic acid was 541 mM.

[0064] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A highly thermostable deoxyadenosine kinase mutant, characterized in that: Compared with the amino acid sequence shown in SEQ ID NO.1, the highly thermostable deoxyadenosine kinase mutant comprises at least one of the following mutations: V40G, D121T and S154L.

2. A nucleic acid, characterized in that The nucleic acid encodes the highly thermostable deoxyadenosine kinase mutant according to claim 1.

3. An expression cassette, characterized in that: The expression cassette comprises the nucleic acid of claim 2.

4. A genetic engineering vector, characterized in that: The genetic engineering vector comprises the nucleic acid according to claim 2 or the expression cassette according to claim 3.

5. The genetic engineering vector according to claim 4, characterized in that: The genetic engineering vector is a eukaryotic expression vector or a prokaryotic expression vector, the eukaryotic expression vector is selected from one or more of pFastBac, pEGFP-N1, pcDNA3.1 and pLVX-CMV-MCS-Linker-EGFP-IRES-Puro, and the prokaryotic expression vector is selected from one or more of pET28a, pET32a, pGEX-4T, pMAL-p2x and pMAL-c2X.

6. The genetic engineering vector according to claim 5, characterized in that: The genetic engineering vector is pET28a(+).

7. A microorganism, characterized in that The microorganism comprises the highly thermostable deoxyadenosine kinase mutant according to claim 1, the nucleic acid according to claim 2, the expression cassette according to claim 3 and / or the genetic engineering vector according to any one of claims 4-6.

8. The microorganism according to claim 7, characterized in that The microorganism is a prokaryotic microorganism or a eukaryotic microorganism. The eukaryotic microorganism is selected from one or more of Saccharomyces cerevisiae, Pichia pastoris and Kluyveromyces marxianus, and the prokaryotic microorganism is selected from one or more of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Salmonella and Streptomyces.

9. The method for preparing a highly thermostable deoxyadenosine kinase mutant according to claim 1, characterized in that: The following steps are involved: (1) Construction of genetic engineering vectors; (2) Perform site-directed mutagenesis of V40G, D121T and / or S154L by PCR to obtain the gene encoding the highly thermostable deoxyadenosine kinase mutant; (3) Expression and purification of highly thermostable deoxyadenosine kinase mutants.

10. The preparation method according to claim 9, characterized in that: In step (2), the primer sequences used for V40G site-directed mutagenesis are shown in SEQ ID NOs. 4-5; the primer sequences used for D121T site-directed mutagenesis are shown in SEQ ID NOs. 18-19; and the primer sequences used for S154L site-directed mutagenesis are shown in SEQ ID NOs. 30-31.

11. Use of the highly thermostable deoxyadenosine kinase mutant according to claim 1, the nucleic acid according to claim 2, the expression cassette according to claim 3, the genetic engineering vector according to any one of claims 4 to 6, and / or the microorganism according to any one of claims 7 to 8 in the preparation of deoxyadenylic acid.

12. A method for preparing deoxyadenylic acid, characterized in that: The method uses the highly thermostable deoxyadenosine kinase mutant of claim 1 and / or the microorganism of any one of claims 7-8 as a catalyst.

13. The method according to claim 12, characterized in that The method uses deoxyadenosine, deoxyadenosine triphosphate and magnesium chloride hexahydrate as substrates, high-thermal stability deoxyadenosine kinase and adenylate kinase as catalysts, and synthesizes deoxyadenylic acid through a one-step reaction.

14. The method according to claim 13, characterized in that The final concentration of deoxyadenosine is 200-400 mM, the final concentration of deoxyadenosine triphosphate is 110-240 mM, the final concentration of magnesium chloride hexahydrate is 50-80 mM, the added amount of high thermal stability deoxyadenosine kinase is 50,000-60,000 U / mL, and the added amount of adenylate kinase is 18,000-25,000 U / mL.

15. The method according to claim 13, characterized in that The one-step reaction is carried out at 37-55°C.

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