Guanylate kinase mutant with improved thermal stability as well as application and product of guanylate kinase mutant

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a guanylate kinase mutant with improved thermal stability and application and a product thereof. Aiming at the defects that wild guanylate kinase is insufficient in thermal stability and easy to inactivate in reaction, mutants (A24Q, S75P, K146P and L172I) are obtained by site-directed mutagenesis modification based on a wild guanylate kinase sequence. The mutant is replaced by specific amino acid residues, so that the thermal stability is remarkably improved, meanwhile, good catalytic activity is kept, wider temperature conditions can be tolerated, and the effective action time of enzyme is prolonged. The mutant can efficiently catalyze a phosphate group transfer reaction of guanylic acid and adenosine triphosphate, and is suitable for enzymatic synthesis of nucleoside compounds such as guanosine diphosphate. The application of the enzyme source can reduce the use amount and supplement frequency of enzyme preparations, reduce the production cost, improve the product yield and quality, and provide reliable enzyme source support for the industrial efficient production of nucleoside products.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a guanylate kinase mutant with improved thermal stability and its applications and products. Background Technology

[0002] Guanylate kinase (GMPK) is a key enzyme in the nucleoside metabolic pathway, catalyzing the phosphate group transfer reaction between guanylate and adenosine triphosphate (ATP) to produce guanosine diphosphate (GDP) and adenosine diphosphate (ATP). This reaction plays an irreplaceable role in energy metabolism and nucleic acid synthesis in organisms. GMPK also has significant applications in industrial biocatalysis, particularly in the green synthesis of nucleoside compounds such as GDP. Compared to chemical synthesis methods, enzymatic catalysis offers significant advantages, including milder reaction conditions, higher specificity, and environmental friendliness, making it an important direction for the industrial production of nucleoside products.

[0003] In existing technologies, guanylate kinases are widely available, including model organisms such as *Saccharomyces cerevisiae*. Among these, some guanylate kinases, due to their well-defined sequences and high feasibility for cloning and expression, have become highly promising enzyme sources for industrial applications. However, wild-type guanylate kinases face a bottleneck in practical applications due to insufficient thermostability. Under normal reaction temperatures or slightly higher temperatures, wild-type guanylate kinases are prone to structural changes leading to inactivation, and their enzyme activity rapidly declines with increasing reaction time. This poor thermostability not only necessitates frequent replenishment of enzyme preparations to maintain reaction efficiency, increasing production costs, but also may lead to incomplete reactions and reduced product yields due to rapid enzyme activity decline, severely limiting its promotion and application in large-scale industrial production.

[0004] Addressing the technical challenge of insufficient thermostability in wild-type guanylate kinase, the development of thermostability-enhanced mutants holds significant theoretical and practical value. From an industrial perspective, thermostability-enhanced guanylate kinase mutants can tolerate a wider range of reaction temperatures, extend enzyme lifespan, and reduce enzyme dosage and replenishment frequency, thereby decreasing labor and material costs in production, improving the production efficiency and product quality of nucleoside compounds, and enhancing the market competitiveness of enzymatic catalysis processes. From an enzyme engineering perspective, screening key mutation sites and constructing thermostable mutants through molecular dynamics simulations and multiple sequence alignment techniques not only clarifies the correlation mechanism between guanylate kinase structure and stability, providing a reference paradigm for the molecular modification of other nucleoside kinases, but also enriches the technical strategies for enzyme thermostability modification, promoting the development of related theories and applications in the field of enzyme engineering. Summary of the Invention

[0005] To address the aforementioned shortcomings, this invention provides a guanylate kinase mutant with significantly improved thermal stability and its applications. This mutant is obtained through site-directed mutagenesis of the wild-type guanylate kinase sequence. By replacing specific amino acid residues, the thermal stability of the enzyme is enhanced, effectively solving the problem that the wild-type enzyme is easily inactivated by temperature during the reaction. It can be efficiently applied to the enzymatic synthesis of nucleoside compounds such as guanosine diphosphate. While maintaining catalytic activity, it improves the temperature tolerance of the reaction system and prolongs the effective action time of the enzyme, providing reliable enzyme source support for the efficient industrial production of nucleoside products.

[0006] The technical solution of this invention is as follows: On one hand, the present invention provides a guanylate kinase mutant with improved thermal stability, the mutant being obtained by site-directed mutagenesis based on wild-type guanylate kinase derived from Saccharomyces cerevisiae, the amino acid sequence of the wild-type guanylate kinase being SEQ ID NO.2, and the mutation site of the mutant being at least one of A24Q, S75P, K146P, and L172I.

[0007] Preferably, in some embodiments, the mutation site of the mutant is A24Q.

[0008] Preferably, in some embodiments, the mutation site of the mutant is S75P.

[0009] Preferably, in some embodiments, the mutation site of the mutant is K146P.

[0010] Preferably, in some embodiments, the mutation site of the mutant is L172I.

[0011] In another aspect, the present invention provides a gene encoding the aforementioned guanylate kinase mutant.

[0012] In another aspect, the present invention provides a recombinant expression vector comprising the aforementioned gene.

[0013] In another aspect, the present invention provides a host cell containing the aforementioned gene or recombinant expression vector.

[0014] Specifically, the host cell can be a prokaryotic host cell or a eukaryotic host cell.

[0015] More specifically, the prokaryotic host cells include, but are not limited to, Escherichia coli, Bacillus, or lactic acid bacteria; the eukaryotic host cells are yeast, filamentous fungi, insect cells, or mammalian cells.

[0016] Preferably, the host cell is Escherichia coli.

[0017] In another aspect, the present invention provides a method for preparing the aforementioned guanylate kinase mutant, comprising culturing and inducing expression in the aforementioned host cells, and obtaining the guanylate kinase mutant after isolation and purification.

[0018] In another aspect, the present invention provides a kit comprising the aforementioned guanylate kinase mutant.

[0019] Specifically, it also includes at least one of adenosine kinase, adenosine triphosphate, buffer solution, and metal ion cofactor.

[0020] Preferably, the metal ion cofactor is magnesium ion, and the buffer solution is at least one of Tris buffer and PBS buffer.

[0021] In another aspect, the present invention provides the application of the aforementioned guanylate kinase mutant, gene, recombinant expression vector as described in claim 3, host cell or kit in catalyzing phosphate group transfer reactions.

[0022] The beneficial effects of this invention are as follows: (1) The thermostability of the mutant is significantly improved, which effectively solves the core defect that wild-type guanylate kinase is easily inactivated by temperature. It can tolerate a wider range of temperature conditions during the reaction, prolong the effective action time of the enzyme, and reduce the adverse effects of enzyme activity decay. (2) While improving thermal stability, the mutant maintains good catalytic activity and can efficiently catalyze the phosphate group transfer reaction between guanosine monophosphate and adenosine triphosphate, ensuring the synthesis efficiency of target products such as guanosine diphosphate. (3) This mutant is suitable for industrial enzymatic production of nucleoside compounds, which can reduce the amount of enzyme preparation used and the frequency of replenishment, reduce the labor and material costs in the production process, and improve product yield and quality. Attached Figure Description

[0023] Figure 1 The thermal stability of guanylate kinase at different temperatures. Detailed Implementation

[0024] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0025] Example 1 Cloning and Expression of Guanylate Kinase Guanylate kinase (nucleotide sequence is...) Saccharomyces cerevisiae S288Cguanylatekinase (GUK1, Gene ID: 852065), according to E. coli Codon preference was used to optimize rare codons in these nucleotide sequences (SEQ ID NO.1). An NcoI restriction site (CCATGG, because the second codon starts with G, only CC is added) was added before the start codon, and an XhoI restriction site was added to the end of the sequence after deleting the stop codon. These sequences were sent to Shanghai Jierui for whole-genome synthesis, and the NcoI / XhoI double restriction site was used to ligate the pET28a(+) vector to construct the pET28a(+)-GMPK expression vector. The constructed plasmid was centrifuged at 10,000 rpm for 10 min, and dissolved in 50 μL of ddH2O using a vortex mixer. After dissolution, it was transformed into *E. coli* BL21(DE3), and the transformed *E. coli* BL21(DE3) was activated and cultured at 37 ℃ and 220 rpm for 1 hour. The cultured plasmid was then plated on LB agar plates containing 50 µg / mL kanamycin and incubated upside down at 37 ℃ overnight. Single colonies were picked from the plate and inoculated into TB liquid medium containing a final concentration of 50 µg / mL kanamycin. Glucose and lactose were added to a final concentration of 5 g / L, and the culture was incubated at 37°C and 220 rpm. 600 The concentration was increased to 0.8-1.2 (preferably 0.9), the temperature was changed to 28 ℃, and the cells were cultured at 220 rpm for 14 h. Then, the cells were centrifuged at 4 ℃ and 8000 rpm for 10 minutes to obtain wet bacterial cells containing guanylate kinase.

[0026] SEQ ID NO.1: ccatgggctcccgtcctatcgtaatttctggcccaagtggtacaggtaaatctacactgttgaagaaattgttcgctgaatacccagattctttcgggtttagtgtttcatccactactcgtaccccacgtgctggcgaagtaaacggtaaggactataactttgtctccgtagatgagttcaaatctatgattaagaacaatgagttcattgaatgggcgcaattctccggtaactactatggtagtactgtcgcttccgtcaaacaagtcagtaaatctggtaagacttgtattttagatattgatatgcagggtgtcaaatctgtcaaggctatcccagagttaaatgcccgctttttgtttattgctccaccatcggtcgaggatttgaaaaaacgtttagaaggtcgtggtacggagaccgaagaatccatcaacaagcgcttaagcgccgctcaagctgaattggcatatgctgagacaggtgcccatgacaaagttattgtcaatgatgatttggacaaggcctacaaggaattgaaggattttatctttgcagaaaaactcgagtaa。

[0027] The amino acid sequence corresponding to SEQ ID NO.1 is shown in SEQ ID NO.3.

[0028] SEQ ID NO.3: [[ID=⑧]]Mgsrpivisgpsgtgkstllkklfaeypdsfgfsvssttrtpragevngkdynfvsvdefksmiknnefiewaqfsgnyygstvasvkqvsksgktcildidmqgvksvkaipelnarflfiappsvedlkkrlegrgteteesinkrlsaaqaelayaetgahdkvivnddldkaykelkdfifaekle.

[0029] Purification of Guanylate Kinase in Example 2 The wet bacterial cells obtained in Example 1 were suspended in buffer A (pH 8.0 containing 0.5 M NaCl, 20 mM imidazole, and 20 mM Tris buffer), sonicated for 15 minutes (ice bath, power 400 W, 3 seconds of disruption, 5 seconds of pause), centrifuged at 4°C and 12000 rpm for 20 minutes, and the supernatant was collected. Protein purification was performed using a Ni affinity column (1.6 × 10 cm, Bio-Rad, USA). The specific procedures were as follows: ① Equilibrate the Ni column with 5 column volumes of binding buffer (pH 8.0 containing 0.5 M NaCl, 20 mM imidazole, and 20 mM Tris buffer) until the baseline stabilized; ② Load the sample at a flow rate of 1 mL / min, with a sample volume of 25-40 mg / mL protein, allowing the target protein to adsorb onto the Ni column; ③ Wash with 6 column volumes of buffer A (pH 8.0 containing 0.5 M NaCl, 20 mM imidazole, and 20 mM Tris buffer) at a flow rate of 1 mL / min until the baseline stabilized; ④ Elute with buffer B (pH 8.0 containing 0.5 M NaCl, 400 mM imidazole, and 20 mM Tris buffer) at a flow rate of 1 mL / min and collect the target protein. The target protein was dialyzed overnight in pH 8.0, 20 mM Tris buffer to obtain purified guanylate kinase. The Ni column was then washed with 5 column volumes of binding buffer (pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl) until the baseline stabilized. The Ni column was then stored in 5 column volumes of ultrapure water containing 20% ​​ethanol. The purified wild-type amino acid sequence is shown in SEQ ID NO.2.

[0030] SEQ ID NO.2: msrpivisgpsgtgkstllkklfaeypdsfgfsvssttrtpragevngkdynfvsvdefksmiknnefiewaqfsgnyygstvasvkqvsksgktcildidmqgvksvkaipelnarflfiappsvedlkkrlegrgteteesinkrlsaaqaelayaetgahdkvivnddldkaykelkdfifaek.

[0031] Example 3: Determination of guanylate kinase activity and thermal stability An enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 µmol of GDP per minute at 30 °C and pH 7.5.

[0032] Enzyme activity detection standard conditions: 8.1425 g / L guanylic acid, 25.615 g / L ATP-Na2, 3.025 g / L Tris, appropriate amount of enzyme solution, react for 15 minutes at 30 ℃ and pH 7.5, sample processing and HPLC detection and analysis.

[0033] HPLC detection conditions for guanosine monophosphate, guanosine diphosphate, adenosine diphosphate, and adenosine triphosphate: InertSustain column TM C18 (4.6 × 150 mm, 5µm, Shimadzu) column, mobile phase acetonitrile:0.1M PBS=5:95, flow rate 1.0 mL / min, detection wavelength 253 nm, injection volume 10 μL, column temperature 25℃.

[0034] Thermostability study of guanylate kinase: Guanylate kinase was diluted 100-fold and placed at 35°C. o C, 40 o C and 50 o C. Enzyme activity was measured by sampling at different times. Results are as follows: Figure 1 As shown: Guanylate kinase at 35 o Under C conditions, the enzyme activity remained at approximately 55% overnight, and at 40°C... o Under C conditions, the enzyme activity remained at approximately 11% overnight, while at 50... o Under C conditions, enzyme activity drops to about 25% of its original value after 1 hour, indicating poor thermal stability.

[0035] Example 4: GMPK molecular dynamics simulation, multiple sequence alignment, and construction of mutant libraries 1. Homology modeling is used to predict the structure of GMPK protein. By inputting the amino acid sequence of GMPK into SWISS-MODEL, the target protein sequence is aligned with the template sequence to construct a preliminary three-dimensional model. The template with the highest GMQE score is selected for modeling to obtain a PDB file containing the three-dimensional structure of GMPK.

[0036] 2. Molecular dynamics (MD) simulations are performed using GROMACS. First, a protein complex file with ligands is prepared after docking with AutoDock Vina. Then, preprocessing is performed by creating a topology file, defining the simulation box, adding solvents and ions, minimizing energy, and balancing NPT and NVT. The parameters to be simulated are modified in the md.mdp file, and the MD simulation is performed to generate a trajectory file. Finally, RMSD and RMSF are generated by inputting commands. RMSD analysis can assess the overall structural stability of the protein during the dynamic simulation, while RMSF analysis helps identify flexible regions within the protein.

[0037] 3. Using the protein blast tool in the NCBI database, with the amino acid sequence of GMPK as a probe, we searched for enzyme proteins highly similar to the GMPK sequence. We selected all amino acid sequences with greater than 40% identity to the GMPK sequence for multiple sequence alignment. Using the ESPript 3.0 tool, we performed multiple sequence alignment on these homologous proteins. By aligning a series of homologous protein sequences, we can identify highly conserved amino acid residues during evolution. These conserved residues are often closely related to protein activity, binding sites, or structural stability. In protein engineering, by purposefully modifying these key residues, we can change the activity, selectivity, or stability of proteins. Combining molecular dynamics simulations and protein multiple sequence alignment methods, we selected important amino acid residue sites to construct a mutant library. The screening principle is as follows: (1) Analyzing RMSF helps to identify flexible regions in protein structure, including the active site of GMPK and relatively active flexible regions. Excluding the relevant active sites and mutating these fluctuating amino acid residues into more rigid amino acid residues may help improve the stability of GMPK.

[0038] (2) Protein multiple sequence alignment can reveal the evolutionary homology of related proteins. It provides key information for understanding the functional regions, active sites, structural domains of proteins and their conservation in different species. It is essential for designing and modifying proteins to improve their activity, stability or change their specific functions. Selecting relatively conserved amino acid residue sites and mutating them into other amino acid residues that occur more frequently in homologous proteins, or mutating them into other amino acid residues with different physicochemical properties from thermophilic microorganisms, is expected to improve the thermal stability of GMPK.

[0039] Based on the above site selection principle, the following 21 single-point mutants were constructed: V6I, K15V, L18I, L18V, L19C, K21A, K21R, A24T, A24Q, S33P, S36Y, A72V, S75F, S75P, S81T, V89T, C96V, S143V, K146P, A150T, and L172I.

[0040] Example 5: Construction, expression, and screening of GMPK mutant libraries 5.1 Construction of GMPK Mutant Library Based on the above site selection principle, the following 21 single-point mutants were constructed: V6I, K15V, L18I, L18V, L19C, K21A, K21R, A24T, A24Q, S33P, S36Y, A72V, S75F, S75P, S81T, V89T, C96V, S143V, K146P, A150T, and L172I. Using the pET28a-GMPK plasmid from Example 1 as a template, and employing the principle of Quick-change site-directed mutagenesis, single-point mutagenesis was first performed using the primers listed in Table 1, mutating the amino acids at each site.

[0041] Table 1 Primer sequence list

[0042] PCR amplification was performed using Takara's KOD high-fidelity polymerase under the following conditions: 95°C. o C 2min, then 55 o C20sec, 72 o C 100sec, 30 loops in total, last 72 seconds o C 10 min. PCR products were recovered from the gel and incubated at 37°C using DpnI enzyme (Fermentas). o Digestion of the gel product under C conditions for 2 hours degraded the initial template. The digestion product was converted to BL21(DE3) and plated onto LB agar plates containing 50 μg / mL kanamycin, incubated at 37°C. o After overnight incubation at C, positive clones were screened and sequenced for verification. Recombinant bacteria with guanylate kinase mutants were obtained.

[0043] 5.2 Expression and Screening of Mutant Engineered Bacteria The crude enzyme solution of the GMPK enzyme mutant was obtained according to the method in Example 1, and the mutant was subjected to treatment at 50 °C according to the method in Example 3. o The thermal stability of A24Q under C conditions was determined, and the results are shown in Table 2: o The half-life of C is 1.51 hours, while that of S75P is 50 hours. o The half-life of C is 2.56 hours, while that of K146P is 50 hours. o The half-life of C is 5.16 h, and that of L172I is 50 h. o The half-life of C is 3.24 h. The above-mentioned crude mutant enzyme solution was purified according to the method in Example 2 to obtain the corresponding pure mutant enzyme solution.

[0044] Table 2

[0045] Application Example 1 A thermostable guanylate kinase mutant was used in the preparation of guanosine diphosphate. Guanosine and adenosine triphosphate (ATP) were used as raw materials in a 100 mL reaction system. The amount of guanosine added was 3.63 g (100 mM), and the amount of ATP added was 7.76 g (110 mM), with a molar ratio of guanosine to ATP of 1:1.1. Magnesium chloride hexahydrate was added at 0.20 g (10 mM), and the pH was 9.0. The amount of purified guanosine kinase A24Q mutant enzyme added was 11,200 U. The reaction was allowed to proceed at 50°C. Samples were taken during the reaction and analyzed by HPLC under the conditions described in Example 3. The final reaction data were: GMP 0.343%, GDP 45.383%, AMP 5.157%, ADP 47.654%, and ATP 1.463%.

[0046] Application Example 2 A thermostable guanylate kinase mutant was used in the preparation of guanosine diphosphate. Guanylate and adenosine triphosphate (ATP) were used as raw materials in a 100 mL reaction system. The amount of guanylate added was 3.63 g (100 mM), and the amount of ATP added was 7.76 g (110 mM), with a molar ratio of guanylate to ATP of 1:1.1. Magnesium chloride hexahydrate was added at 0.20 g (10 mM), and the pH was 9.0. The amount of purified guanylate kinase S75P mutant enzyme added was 0.96 million U. The reaction was allowed to proceed at 50°C. Samples were taken during the reaction and analyzed by HPLC under the conditions described in Example 3. The final reaction data were: GMP 0.567%, GDP 45.143%, AMP 5.026%, ADP 47.727%, and ATP 1.537%.

[0047] Application Example 3 A thermostable guanylate kinase mutant was used in the preparation of guanosine diphosphate. Guanylate and adenosine triphosphate (ATP) were used as raw materials in a 100 mL reaction system. The amount of guanylate added was 7.26 g (200 mM), and the amount of ATP added was 15.52 g (220 mM), with a molar ratio of guanylate to ATP of 1:1.1. Magnesium chloride hexahydrate was added at 0.20 g (10 mM), and the pH was 9.5. The amount of purified guanylate kinase L172I mutant enzyme added was 18,300 U. The reaction was allowed to proceed at 50°C. Samples were taken during the reaction and analyzed by HPLC under the conditions described in Example 3. The final reaction data were: GMP 0.863%, GDP 44.847%, AMP 5.432%, ADP 52.665%, and ATP 1.093%.

[0048] Application Example 4 A thermostable guanylate kinase mutant was used in the preparation of guanosine diphosphate. Guanylate and adenosine triphosphate (ATP) were used as raw materials in a 100 mL reaction system. The amount of guanylate added was 7.26 g (200 mM), and the amount of ATP added was 15.52 g (220 mM), with a molar ratio of guanylate to ATP of 1:1.1. Magnesium chloride hexahydrate was added at 0.20 g (10 mM), and the pH was 9.5. The amount of purified guanylate kinase K146P mutant enzyme added was 11,200 U. The reaction was allowed to proceed at 50°C. Samples were taken during the reaction and analyzed by HPLC under the conditions described in Example 3. The final reaction data were: GMP 1.412%, GDP 44.314%, AMP 7.156%, ADP 46.695%, and ATP 0.423%.

[0049] Application Example 5 A thermostable guanylate kinase mutant was used in the preparation of guanosine diphosphate. Guanylate and adenosine triphosphate (ATP) were used as raw materials in a 100 mL reaction system. The amount of guanylate added was 7.26 g (200 mM), and the amount of ATP added was 7.76 g (220 mM). The molar ratio of guanylate to ATP was 1:1.1. Magnesium chloride hexahydrate was added at 0.20 g (10 mM), and the pH was 9.5. The amount of purified guanylate kinase L172I mutant enzyme added was 18,300 U, and the amount of adenosine kinase added was 12,100 U. The reaction was allowed to proceed at 50°C. Samples were taken during the reaction and analyzed by HPLC under the conditions described in Example 3. The final reaction data were: GMP 1.263%, GDP 71.600%, AMP 25.680%, ADP 1.231%, and ATP 0.226%.

[0050] Application Example 6 A thermostable guanylate kinase mutant was used in the preparation of guanosine diphosphate. Guanylate and adenosine triphosphate (ATP) were used as raw materials in a 100 mL reaction system. The amount of guanylate added was 7.26 g (200 mM), and the amount of ATP added was 7.76 g (110 mM). The molar ratio of guanylate to ATP was 2:1.1. Magnesium chloride hexahydrate was added at 0.20 g (10 mM), and the pH was 9.5. The amount of purified guanylate kinase K146P mutant enzyme added was 11,200 U, and the amount of adenosine kinase added was 12,100 U. The reaction was allowed to proceed at 50°C. Samples were taken during the reaction and analyzed by HPLC under the conditions described in Example 3. The final reaction data were: GMP 1.653%, GDP 71.210%, AMP 25.452%, ADP 1.432%, and ATP 0.253%.

[0051] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A guanylate kinase mutant with improved thermal stability, characterized in that, The guanylate kinase mutant was obtained by site-directed mutagenesis based on wild-type guanylate kinase from Saccharomyces cerevisiae. The amino acid sequence of the wild-type guanylate kinase is SEQ ID NO.

2. The mutation site of the mutant is at least one of A24Q, S75P, K146P, and L172I.

2. A gene encoding the guanylate kinase mutant of claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the gene as described in claim 2.

4. A host cell, characterized in that, The host cell contains the gene of claim 2 or the recombinant expression vector of claim 3.

5. The host cell according to claim 4, characterized in that, The host cell is a prokaryotic host cell or a eukaryotic host cell.

6. The host cell according to claim 5, characterized in that, The prokaryotic host cell is Escherichia coli, Bacillus, or lactic acid bacteria; the eukaryotic host cell is yeast, filamentous fungi, insect cells, or mammalian cells.

7. A method for preparing the guanylate kinase mutant according to claim 1, characterized in that, This includes culturing and inducing expression in the host cells described in any one of claims 4-6, and then separating and purifying the guanylate kinase mutant.

8. A reagent kit, characterized in that, Includes the guanylate kinase mutant of claim 1.

9. The reagent kit according to claim 8, characterized in that, It also contains at least one of adenosine kinase, adenosine triphosphate, buffer solution, and metal ion cofactor.

10. The use of the guanylate kinase mutant of claim 1, the gene of claim 2, the recombinant expression vector of claim 3, the host cell of any one of claims 4-6, or the kit of claim 8 or 9 in catalyzing phosphate group transfer reactions.

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