An aminoglycoside 2'-phosphotransferase mutant and its application

Abstract

The present invention discloses an aminoglycoside 2'-phosphotransferase mutant and its application, relating to the field of biotransformation technology, including the following method for preparing the aminoglycoside 2'-phosphotransferase mutant: S1, cloning and expressing the aminoglycoside 2'-phosphotransferase; S2, purification of the aminoglycoside 2'-phosphotransferase; S3, determination of the enzyme activity and substrate specificity of the aminoglycoside 2'-phosphotransferase; S4, construction of a single-point mutant; S5, construction, expression and screening of an AphIf mutant library. The present invention obtains an aminoglycoside 2'-phosphotransferase mutant capable of specifically degrading GTP without degrading UTP through cloning, expression, purification and screening, which can solve the problems of difficult separation and purification of GTP in the UTP production process, high GTP impurity content in the UTP production process, and low UTP yield and purity.

Description

Technical Field

[0001] The present invention relates to the technical field of biotransformation, in particular to an aminoglycoside 2'-phosphotransferase mutant and application thereof. Background Art

[0002] Traditional transferase mutants cannot effectively solve the problem of difficult separation and purification of GTP. This is mainly because GTP and UTP are very similar in chemical properties, making it difficult for traditional separation methods to effectively distinguish them. In addition, traditional transferase mutants cannot effectively reduce the content of GTP impurities in the UTP production process, resulting in serious impact on the purity of the UTP product. Ultimately, these problems jointly lead to generally low yield and purity of UTP, limiting the possibility of its large-scale industrial production. The transferase mutants of the present application can solve the problems of difficult separation and purification of GTP in the UTP production process, high GTP impurity content in the UTP production process, and low UTP yield and purity.

[0003] The defects of existing transferase mutants are:

[0004] Patent document US10519429B2 discloses a nicotinamide phosphoribosyltransferase mutant and its application. This document mainly considers how to provide a NAMPT mutant with higher catalytic activity than the conventional wild-type parent, but does not consider how to solve the problems of difficult separation and purification of GTP during UTP production, high GTP impurity content during UTP production, and low UTP yield and purity.

[0005] Patent document CN109295027B discloses a glycosyltransferase mutant. This document mainly considers how to improve the catalytic activity of the glycosyl transfer reaction, but does not consider how to improve the expression level and activity of the enzyme mutant and realize the industrial production of the enzyme.

[0006] Patent document CN112080480B discloses a glycosyltransferase mutant and its application. This document mainly considers how to increase the accumulation of specific steviol glycoside intermediates, but does not consider how to improve enzyme purity, optimize purification conditions, enhance enzyme activity and stability, achieve large-scale enzyme purification, and reduce purification costs.

[0007] Patent document CN118207179B discloses a glycosyltransferase mutant and its application. The document mainly considers how to solve the problem of low glycosyltransferase activity in the existing technology, but does not consider how to solve the problems of accurate determination of enzyme activity and substrate specificity, design and screening of enzyme mutants based on structural information, and performance optimization of enzyme mutants. Summary of the Invention

[0008] The object of the present invention is to provide an aminoglycoside 2'-phosphotransferase mutant and its application to solve the problems raised in the above background technology.

[0009] To achieve the above object, the present invention provides the following technical solution: an aminoglycoside 2'-phosphotransferase mutant, and a method for preparing the aminoglycoside 2'-phosphotransferase mutant is as follows:

[0010] S1, cloning and expression of aminoglycoside 2'-phosphotransferase;

[0011] S2, purification of aminoglycoside 2'-phosphotransferase;

[0012] S3, aminoglycoside 2'-phosphotransferase enzyme activity and substrate specificity determination;

[0013] S4. Based on the known aminoglycoside 2'-phosphotransferase structure information, modeling and molecular docking were performed on its homologous protein AphIf to construct single-point mutants;

[0014] S5, construction, expression, and screening of AphIf mutant library;

[0015] In S2, the following is also included:

[0016] S21, suspend the wet cells obtained in S1 in buffer A, disrupt with ultrasound, and centrifuge at 4°C and 12,000 rpm for 20 min. Collect the supernatant to obtain a crude extract;

[0017] S22. Use Ni affinity column to purify protein. The specific operations are as follows: ① Equilibrate the Ni column with 5 column volumes of buffer A until the baseline is stable; ② Load the crude extract sample at a flow rate of 1 mL / min, with a loading amount of 25-40 mg / mL protein, to allow the target protein to adsorb on the Ni column; ③ Wash the impurities with 6 column volumes of buffer A at a flow rate of 1 mL / min until the baseline is stable; ④ Elute with buffer B at a flow rate of 1 mL / min to collect the target protein, and dialyze the target protein in pH 8.0, 20 mM Tris buffer overnight to obtain purified nicotinamide phosphoribosyltransferase; ⑤ Wash the Ni column with 5 column volumes of buffer A until the baseline is stable, and preserve the Ni column with 5 column volumes of ultrapure water containing 20% ethanol.

[0018] Preferably, ultrasonication is carried out in an ice bath environment with a power of 400 W and a working cycle of 3 seconds of ultrasonication and 5 seconds of pause for 15 minutes;

[0019] Buffer A was 20 mM Tris buffer at pH 8.0 containing 0.5 M NaCl and 20 mM imidazole;

[0020] Buffer B was pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 400 mM imidazole;

[0021] The Ni affinity column has a size of 1.6 × 10 cm and is manufactured in the United States.

[0022] Preferably, in S1, the following is further included:

[0023] S11. The aminoglycoside 2'-phosphotransferase gene was optimized for rare codons in these nucleotide sequences according to the codon preference of E. coli, an Nco I restriction site was added before the start codon, the stop codon was deleted, and an Xho I restriction site was added at the end of the sequence;

[0024] S12. These sequences were sent to Shanghai Jierui for full gene synthesis and ligated into the pET28a(+) vector using the NcoⅠ / XhoⅠ double restriction sites to construct the pET28a(+)-AphIf expression vector, i.e., the pET28a-AphIf plasmid;

[0025] S13. The constructed pET28a-AphIf plasmid was collected by centrifugation at 10,000 rpm for 10 min to precipitate, and 50 μL of ddH2O was added to dissolve the plasmid on a vortex shaker. After dissolution, E. coli BL21 (DE3) was transformed and activated. The transformed E. coli BL21 (DE3) was cultured at 37°C and 220 rpm for 1 hour, and then spread on an LB plate containing 50 μg / mL kanamycin resistance and cultured in an inverted manner at 37°C overnight.

[0026] S14. Pick a single colony from the plate and inoculate it into TB liquid culture medium containing a final concentration of 50µg / mL kanamycin. Add glucose and 20g / L lactose at a final concentration of 5g / L. Culture at 37°C and 220rpm. When the OD600 reaches 0.8-1.2, change the temperature to 28°C and continue to culture at 220rpm for 14h. Finally, centrifuge at 4°C and 8000rpm for 10 minutes to obtain wet bacteria containing aminoglycoside 2'-phosphotransferase.

[0027] Preferably, the nucleotide sequence of the aminoglycoside 2'-phosphotransferase gene is the 9696-10589 sequence of Campylobacter jejuniplasmidpCG8245 GenBank: AY701528.1, and the amino acid sequence Genbank is AAW34150.1;

[0028] The NcoⅠ restriction site is CCATGG.

[0029] Preferably, S3 further includes the following:

[0030] S31. Standard conditions for enzyme activity assay are: substrate 25 g / L L-GTP, inhibitor 25 g / L kanamycin sulfate, cofactor 2.5 g / L MgCl₂∙6H₂O, appropriate amount of enzyme solution, reaction at 35°C and pH 8.0 for 20 minutes, sample preparation and HPLC analysis.

[0031] Liquid phase detection conditions for S32, GDP, and GTP include the InertSustain column TM C18, mobile phase is acetonitrile:0.04MPBS=7:93, flow rate is 1.0mL / min, detection wavelength is 262nm, injection volume is 10μL, column temperature is 40℃.

[0032] Preferably, InertSustain TM The size of C18 is 4.6×150mm, the particle size is 5µm, and the brand is Shimadzu;

[0033] The specificity of aminoglycoside 2'-phosphotransferase was determined by replacing GTP with ATP and UTP in the standard conditions of the enzymatic assay.

[0034] Preferably, in S4, the following is further included:

[0035] S41. Using homology modeling, a preliminary three-dimensional model of AphIf was constructed in SWISS-MODEL based on the alignment of the AphIf amino acid sequence with a highly homologous template sequence, and finally a three-dimensional model of AphIf was obtained;

[0036] S42. Using the flexible docking principle, during the molecular docking process, the conformational changes of the ligand GTP are taken into account, and the amino acid residues near the active site are allowed to be flexible. After removing water molecules, adding hydrogen atoms, setting the pH, and determining the docking box range, the binding energy and interaction force between the ligand and protein are analyzed to select the most suitable docking model;

[0037] S43. Protein three-dimensional structure analysis was performed using PyMOL, combined with HotSpotWizard analysis of "hotspot residues" to construct a mutation library. The screening criteria included selecting amino acid residues that interact with GTP near the 5Å region of GTP in the optimal docking model, automatically identifying mutation hotspots using HotSpotWizard, and prioritizing "hotspot" amino acids near the 5Å region of GTP while excluding active sites. Based on this, the following 20 single-point mutants were constructed:

[0038] S27C, S43R, K47S, K48E, G49T, Y50L, Y50Q, K52N, E80D, I130Y, T134L, I135K, N137D, E144D, E150K, I176V, H192N, S210F, I220L, Q254A.

[0039] Preferably, in S5, the following is further included:

[0040] S51. Using the pET28a-AphIf plasmid constructed in S1 as a template, single-point mutants were mutated using the Quick-change mutagenesis method and primers to construct a mutation library;

[0041] S52. PCR amplification was performed using Takara's KOD high-fidelity polymerase according to the amplification conditions. The PCR product was then recovered from the gel and digested with DpnI to degrade the initial template. The digested product was transformed into BL21 (DE3) and positive clones were screened on plates and sequenced to obtain recombinant bacteria with the AphIf enzyme mutant.

[0042] S53. Prepare mutant wet cells according to S1, determine the mutant substrate specificity according to S3, and then purify the crude enzyme solution prepared from the mutant wet cells according to S2 to obtain mutant pure enzyme solution.

[0043] Preferably, 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.

[0044] Preferably, the aminoglycoside 2'-phosphotransferase mutant is used as follows: the aminoglycoside 2'-phosphotransferase mutant is used to specifically degrade GTP into GDP, and then GDP is removed by utilizing the adsorption difference between GDP and UTP on an anion exchange column, thereby obtaining high-purity UTP.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] 1. The present invention obtains an aminoglycoside 2'-phosphotransferase mutant that can specifically degrade GTP without degrading UTP through cloning, expression, purification and screening. UTP is usually converted using whole-cell catalysis of Saccharomyces cerevisiae. However, since Saccharomyces cerevisiae is a living cell, it carries the impurity GTP. The properties of UTP and GTP are very similar, making it difficult to separate and purify them, resulting in an extremely low yield. The aminoglycoside 2'-phosphotransferase mutant has high substrate specificity and can efficiently convert GTP into GDP without degrading UTP. In the production process of UTP, the enzyme mutant is introduced. The method specifically degrades GTP, thereby reducing the content of GTP in the UTP product. At the same time, due to the difference in adsorption between GDP and UTP on the anion exchange column, GDP can be separated from UTP by the anion exchange column, further improving the purity of UTP. Through the above steps, the present invention not only effectively controls the limit of GTP, but also greatly improves the yield and purity of UTP, making it more suitable for large-scale industrial production. Therefore, the problems of difficult separation and purification of GTP in the UTP production process, high impurity content of GTP in the UTP production process, and low yield and purity of UTP can be solved.

[0047] 2. The present invention improves the expression level of the enzyme in E. coli BL21 (DE3) by codon optimization of the aminoglycoside 2'-phosphotransferase gene and adjusting the nucleotide sequence according to the codon preference of E. coli BL21 (DE3). By adding appropriate restriction sites and connecting to the efficient pET28a-AphIf expression vector, the efficient expression of the enzyme is further ensured. At the same time, the present invention provides the possibility for industrial production of the enzyme by constructing the pET28a-AphIf expression vector and efficiently expressing the aminoglycoside 2'-phosphotransferase mutant in E. coli BL21 (DE3). By optimizing the culture conditions, high yield is obtained. The enzyme-containing wet bacteria lay the foundation for subsequent enzyme purification and application. The substrate specificity and catalytic efficiency of the enzyme mutants obtained by the method of the present invention may be optimized, thereby improving the efficiency of biotransformation and product purity. Aminoglycoside antibiotics are an important class of antibacterial drugs, but the emergence of bacterial resistance to them is a serious problem. Aminoglycoside 2'-phosphotransferase is a mechanism for bacteria to develop resistance, and its antibacterial activity is reduced by phosphorylation modification of antibiotic molecules. The present invention may help understand the resistance mechanism by preparing and screening enzyme mutants, and provide clues for the development of new antibiotics or inhibitors. Therefore, it can solve the problem of how to improve the expression level and activity of enzyme mutants and realize the industrial production of enzymes.

[0048] 3. The present invention adopts Ni affinity column purification technology to effectively separate the target protein - aminoglycoside 2'-phosphotransferase from the crude extract. After the steps of equilibration with buffer A, loading of the crude extract, washing of impurities and elution with buffer B, high-purity aminoglycoside 2'-phosphotransferase is finally obtained. During the purification process, the present invention optimizes the conditions of the steps of ultrasonic fragmentation, centrifugation, loading, washing and elution. Ultrasonic fragmentation is carried out in an ice bath environment to avoid damage to the activity of the enzyme caused by high temperature. The working cycle is set to fragmentation for 3 seconds and pause for 5 seconds, which lasts for 15 minutes to ensure the fragmentation effect while reducing energy consumption. The centrifugation conditions, loading amount, washing and elution flow rates are also aimed at improving purification efficiency and purity. The purification method provided by the present invention is not only suitable for small-scale purification on a laboratory scale, but can also be used for large-scale purification in industrial production. By adjusting parameters such as the size of the Ni affinity column, the sample loading amount, and the volume of the buffer, the production needs of different scales can be met. Moreover, as a commonly used protein purification technology, the Ni affinity column has the advantages of simple operation and relatively low cost. By optimizing the purification steps and conditions, the purification cost is further reduced and the economic benefit is improved. The purified aminoglycoside 2'-phosphotransferase can be used in multiple fields such as biotransformation, drug synthesis, and enzyme engineering. Therefore, the problems of improving the purity of the enzyme, optimizing the purification conditions, improving the activity and stability of the enzyme, realizing large-scale purification of the enzyme, and reducing the purification cost can be solved.

[0049] 4. The present invention provides standard conditions for detecting the enzyme activity of aminoglycoside 2'-phosphotransferase, including key parameters such as substrate concentration, inhibitor concentration, cofactor concentration, reaction temperature, pH value, and reaction time, thereby ensuring the accuracy and repeatability of enzyme activity determination. Through HPLC detection and analysis, the amount of enzyme reaction product generated can be accurately determined, thereby evaluating the activity level of the enzyme. At the same time, the present invention also determines the substrate specificity of aminoglycoside 2'-phosphotransferase by replacing the substrate GTP with ATP and UTP, providing important information for understanding the catalytic mechanism and substrate selectivity of the enzyme. Using the homology modeling method, the present invention successfully constructed a three-dimensional model of the aminoglycoside 2'-phosphotransferase homologous protein AphIf, which provides information for subsequent molecular docking and mutant design. The invention provides a basis. Through the principle of flexible docking, the conformational changes of the ligand GTP and the flexibility of the amino acid residues near the active site are taken into account, thereby improving the accuracy and reliability of molecular docking. Combined with the analysis of "hotspot residues" by HotSpotWizard, the invention screens out amino acid residues that interact with GTP and constructs a mutation library containing 20 single-point mutants, providing rich candidate materials for subsequent enzyme mutant screening and performance optimization. By screening the mutation library, the invention obtains aminoglycoside 2'-phosphotransferase mutants with higher enzyme activity, stronger substrate specificity or better stability. Therefore, the problems of accurate determination of enzyme activity and substrate specificity, design and screening of enzyme mutants based on structural information, and performance optimization of enzyme mutants can be solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is a three-dimensional diagram and active center site diagram of the aminoglycoside 2'-phosphotransferase of the present invention. DETAILED DESCRIPTION

[0051] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention. Example 1

[0052] See also Figure 1 The present invention provides an embodiment: an aminoglycoside 2'-phosphotransferase mutant. The preparation method of the aminoglycoside 2'-phosphotransferase mutant is as follows:

[0053] S1, cloning and expression of aminoglycoside 2'-phosphotransferase;

[0054] In S1, the following is also included:

[0055] S11. The aminoglycoside 2'-phosphotransferase gene was optimized for rare codons in these nucleotide sequences according to the codon preference of E. coli, an Nco I restriction site was added before the start codon, the stop codon was deleted, and an Xho I restriction site was added at the end of the sequence;

[0056] S12. These sequences were sent to Shanghai Jierui for full gene synthesis and ligated into the pET28a(+) vector using the NcoⅠ / XhoⅠ double restriction sites to construct the pET28a(+)-AphIf expression vector, i.e., the pET28a-AphIf plasmid;

[0057] S13. The constructed pET28a-AphIf plasmid was collected by centrifugation at 10,000 rpm for 10 min to precipitate, and 50 μL of ddH2O was added to dissolve the plasmid on a vortex shaker. After dissolution, E. coli BL21 (DE3) was transformed and activated. The transformed E. coli BL21 (DE3) was cultured at 37°C and 220 rpm for 1 hour, and then spread on an LB plate containing 50 μg / mL kanamycin resistance and cultured in an inverted manner at 37°C overnight.

[0058] S14. Pick a single colony from the plate and inoculate it into TB liquid medium containing a final concentration of 50µg / mL kanamycin, add 5g / L glucose and 20g / L lactose to a final concentration, and culture at 37°C and 220rpm. When the OD 600 When the concentration reached 0.8-1.2, the temperature was changed to 28°C, and the culture was continued at 220 rpm for 14 h. Finally, the culture was centrifuged at 4°C and 8000 rpm for 10 min to obtain wet cells containing aminoglycoside 2'-phosphotransferase.

[0059] The nucleotide sequence of the aminoglycoside 2'-phosphotransferase gene is 9696-10589 of Campylobacter jejuniplasmidpCG8245 GenBank: AY701528.1, and the amino acid sequence Genbank is AAW34150.1;

[0060] The NcoⅠ restriction site is CCATGG. Example 2

[0061] An aminoglycoside 2'-phosphotransferase mutant. The preparation method of the aminoglycoside 2'-phosphotransferase mutant is as follows:

[0062] S2, purification of aminoglycoside 2'-phosphotransferase;

[0063] In S2, the following is also included:

[0064] S21, suspend the wet cells obtained in S1 in buffer A, disrupt with ultrasound, and centrifuge at 4°C and 12,000 rpm for 20 min. Collect the supernatant to obtain a crude extract;

[0065] S22. Use Ni affinity column to purify protein. The specific operations are as follows: ① Equilibrate the Ni column with 5 column volumes of buffer A until the baseline is stable; ② Load the crude extract sample at a flow rate of 1 mL / min, with a loading amount of 25-40 mg / mL protein, to allow the target protein to adsorb on the Ni column; ③ Wash the impurities with 6 column volumes of buffer A at a flow rate of 1 mL / min until the baseline is stable; ④ Elute with buffer B at a flow rate of 1 mL / min to collect the target protein, and dialyze the target protein in pH 8.0, 20 mM Tris buffer overnight to obtain purified nicotinamide phosphoribosyltransferase; ⑤ Wash the Ni column with 5 column volumes of buffer A until the baseline is stable, and preserve the Ni column with 5 column volumes of ultrapure water containing 20% ethanol.

[0066] Ultrasonic disruption was performed in an ice bath environment with a power of 400 W and a working cycle of 3 seconds of disruption and 5 seconds of pause for 15 minutes;

[0067] Buffer A was 20 mM Tris buffer at pH 8.0 containing 0.5 M NaCl and 20 mM imidazole;

[0068] Buffer B was pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 400 mM imidazole;

[0069] The Ni affinity column has a size of 1.6 × 10 cm and is manufactured in the United States. Example 3

[0070] An aminoglycoside 2'-phosphotransferase mutant. The preparation method of the aminoglycoside 2'-phosphotransferase mutant is as follows:

[0071] S3, aminoglycoside 2'-phosphotransferase enzyme activity and substrate specificity determination;

[0072] In S3, it also includes the following:

[0073] S31. Standard conditions for enzyme activity assay are: substrate 25 g / L L-GTP, inhibitor 25 g / L kanamycin sulfate, cofactor 2.5 g / L MgCl₂∙6H₂O, appropriate amount of enzyme solution, reaction at 35°C and pH 8.0 for 20 minutes, sample preparation and HPLC analysis.

[0074] Liquid phase detection conditions for S32, GDP, and GTP include the InertSustain column TMC18, mobile phase is acetonitrile:0.04MPBS=7:93, flow rate is 1.0mL / min, detection wavelength is 262nm, injection volume is 10μL, column temperature is 40℃.

[0075] InertSustain TM The size of C18 is 4.6×150mm, the particle size is 5µm, and the brand is Shimadzu;

[0076] The specificity of aminoglycoside 2'-phosphotransferase was determined by replacing GTP with ATP and UTP in the standard conditions of the enzymatic assay.

[0077] Furthermore, the measurement results are as follows:

[0078]

[0079] After determination, the enzyme activity of the purified aminoglycoside 2'-phosphotransferase was 79 U / mL, the enzyme activity against ATP was 4.63 U / mL, and the enzyme activity relative to GTP was only 5.86%. The enzyme activity against UTP was 11.29 U / mL, and the enzyme activity relative to GTP was only 14.29%. Example 4

[0080] An aminoglycoside 2'-phosphotransferase mutant. The preparation method of the aminoglycoside 2'-phosphotransferase mutant is as follows:

[0081] S4. Based on the known aminoglycoside 2'-phosphotransferase structure information, modeling and molecular docking were performed on its homologous protein AphIf to construct single-point mutants;

[0082] In S4, it also includes the following:

[0083] S41. Using homology modeling, a preliminary three-dimensional model of AphIf was constructed in SWISS-MODEL based on the alignment of the AphIf amino acid sequence with a highly homologous template sequence, and finally a three-dimensional model of AphIf was obtained;

[0084] S42. Using the flexible docking principle, during the molecular docking process, the conformational changes of the ligand GTP are taken into account, and the amino acid residues near the active site are allowed to be flexible. After removing water molecules, adding hydrogen atoms, setting the pH, and determining the docking box range, the binding energy and interaction force between the ligand and protein are analyzed to select the most suitable docking model;

[0085] S43. Protein three-dimensional structure analysis was performed using PyMOL, combined with HotSpotWizard analysis of "hotspot residues" to construct a mutation library. The screening criteria included selecting amino acid residues that interact with GTP near the 5Å region of GTP in the optimal docking model, automatically identifying mutation hotspots using HotSpotWizard, and prioritizing "hotspot" amino acids near the 5Å region of GTP while excluding active sites. Based on this, the following 20 single-point mutants were constructed:

[0086] S27C, S43R, K47S, K48E, G49T, Y50L, Y50Q, K52N, E80D, I130Y, T134L, I135K, N137D, E144D, E150K, I176V, H192N, S210F, I220L, Q254A.

[0087] Furthermore, the homology modeling method was used to predict the structure of AphIf protein. By inputting the amino acid sequence of AphIf into SWISS-MODEL, the target protein sequence was aligned with the template sequence, and a preliminary three-dimensional model was constructed. The template sequence with the highest homology to the amino acid sequence of AphIf was selected, and finally the three-dimensional model of AphIf was obtained, which is Figure 1 .

[0088] Using the principle of flexible docking and AutoDockVina, docking is performed, allowing the conformational changes of the ligand, small molecule GTP, and amino acid residues near the active site to occur during the docking process, thereby increasing the accuracy of the predicted docking model. Water molecules are removed from the small molecule ligand and the large protein, hydrogen atoms are added, and the environmental pH is set. Based on the active site, the docking box range is determined, and the docking file and docking script are specified. Finally, the binding energy and related forces between the ligand and the protein are analyzed to select the optimal docking model.

[0089] The three-dimensional structure of the protein was analyzed by PyMOL, and the "hotspot residues" in the protein were analyzed by HotSpotWizard (an online tool based on semi-rational design). The mutation library was further constructed, and the screening conditions were determined as follows:

[0090] (1) The most suitable docking model was selected, its crystal structure was analyzed by PyMOL, and the amino acid residues near the 5Å of the small molecule ligand GTP were selected, especially the sites that interact with the GTP small molecule, which may affect the substrate preference of AphIf.

[0091] (2) Use HotSpotWizard to automatically identify mutation hotspots. By integrating structural, functional, and evolutionary information, we select hotspot positions suitable for mutagenesis. These hotspots are crucial for improving protein stability, catalytic activity, substrate specificity, and enantioselectivity. After importing the AphIf protein model, the server automatically identifies hotspots suitable for mutagenesis. After the calculation is completed, the relevant hotspots and functional residues will be highlighted. Hotspot amino acid residues with non-essential, catalytic pocket, and substrate channel labels are preferentially selected for mutation.

[0092] (3) Combining the above two screening methods, the “hotspot” amino acids near GTP5Å should be preferentially selected. At the same time, the mutation site should exclude the active site of AphIf, and the introduced mutation should generate new interaction forces with the ligand small molecule to improve substrate affinity.

[0093] Based on the above screening principle, the following 20 single-point mutants were constructed: S27C, S43R, K47S, K48E, G49T, Y50L, Y50Q, K52N, E80D, I130Y, T134L, I135K, N137D, E144D, E150K, I176V, H192N, S210F, I220L, and Q254A. Example 5

[0094] An aminoglycoside 2'-phosphotransferase mutant. The preparation method of the aminoglycoside 2'-phosphotransferase mutant is as follows:

[0095] S5, construction, expression, and screening of AphIf mutant library;

[0096] In S5, it also includes the following:

[0097] S51. Using the pET28a-AphIf plasmid constructed in S1 as a template, single-point mutants were mutated using the Quick-change mutagenesis method and primers to construct a mutation library;

[0098] S52. PCR amplification was performed using Takara's KOD high-fidelity polymerase according to the amplification conditions. The PCR product was then recovered from the gel and digested with DpnI to degrade the initial template. The digested product was transformed into BL21 (DE3) and positive clones were screened on plates and sequenced to obtain recombinant bacteria with the AphIf enzyme mutant.

[0099] S53. Prepare mutant wet cells according to S1, determine the mutant substrate specificity according to S3, and then purify the crude enzyme solution prepared from the mutant wet cells according to S2 to obtain mutant pure enzyme solution.

[0100] The amplification conditions were: 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.

[0101] Furthermore, the pET28a-AphIf plasmid in S1 was used as a template, the Quick-change mutagenesis method was used, and primers were used to mutate the amino acids at each site to construct a mutation library.

[0102]

[0103]

[0104] PCR amplification used Takara's KOD high-fidelity polymerase, and the amplification conditions were: 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;

[0105] The PCR product was recovered from the gel and digested with DpnI enzyme at 37°C for 2 h to degrade the initial template. The digestion product was transformed into BL21 (DE3) and spread onto LB agar plates containing 50 μg / mL kanamycin. The plates were cultured at 37°C overnight, and positive clones were screened and verified by sequencing to obtain recombinant bacteria with AphIf enzyme mutants.

[0106] The crude enzyme solution of the AphIf enzyme mutant was obtained according to the method of S1, and the substrate specificity of the mutant was determined according to the method of S3. The results are as follows:

[0107]

[0108] The results showed that the activity of I220L with GTP as substrate was 20.67 U / mL, while the activity with UTP as substrate was only 0.84% of that with GTP as substrate, which greatly improved the substrate specificity.

[0109] The activity of K48E with UTP as substrate is 1.66% of that with GTP as substrate, the activity of Y50Q with UTP as substrate is 3.64% of that with GTP as substrate, the activity of K47S with UTP as substrate is 5.47% of that with GTP as substrate, and the activity of K52N with UTP as substrate is 6.78% of that with GTP as substrate, indicating that the substrate specificity has been improved to varying degrees.

[0110] The above mutant crude enzyme solution was purified according to the method of S2 to obtain the corresponding mutant pure enzyme solution. Example 6

[0111] The aminoglycoside 2'-phosphotransferase mutant is used as follows: GTP is specifically degraded into GDP by the aminoglycoside 2'-phosphotransferase mutant, and GDP is then removed by utilizing the difference in adsorption of GDP and UTP by an anion exchange column, thereby obtaining high-purity UTP.

[0112] Furthermore, aminoglycoside 2'-phosphotransferase mutants were used to remove GTP from UTP:

[0113] The crude UTP product had a UTP purity of 92.27%, with impurities of 4.95% GTP and 1.37% UDP. According to the description in S2, a 1.5-L reaction system was prepared by adding 113.5 g crude UTP (undried), 7.50 g kanamycin sulfate, and 3.05 g magnesium chloride hexahydrate. The pH was adjusted to 8.75 with the addition of NaOH solution. After saturation, the A262 nm reading was 1055. 228.76 U of pure AphIf mutant enzyme was added. The reaction was incubated in a 35°C waterbath. After 9 h of reaction, the residual GTP content was 0.106% and the UTP content was 92.651%.

[0114] After separation on an ion exchange column, the purity of the obtained UTP wet powder was 97.522%, and that of GTP was 0.092%. After drying, the purity of UTP was 95.513%, and that of GTP was 0.089%. Example 7

[0115] Removal of GTP from UTP using aminoglycoside 2'-phosphotransferase mutants:

[0116] The crude UTP product had a UTP purity of 92.27%, with impurities of 4.95% GTP and 1.37% UDP. According to S2, a 1.5-L reaction system was prepared by adding 113.5 g crude UTP (undried), 7.50 g streptomycin, and 3.05 g magnesium chloride hexahydrate. The pH was adjusted to 8.75 with NaOH solution. After saturation, the A262nm value was 1026. 228.76 U of pure AphIf mutant enzyme was added. The reaction was incubated in a 35°C waterbath. After 11 h, the residual GTP content was 0.131% and the UTP content was 92.643%.

[0117] After separation on an ion exchange column and drying, the purity of UTP was 96.074% and that of GTP was 0.115%. Example 8

[0118] Removal of GTP from UTP using aminoglycoside 2'-phosphotransferase mutants:

[0119] The crude UTP product had a UTP purity of 92.27%, with impurities of 4.95% GTP and 1.37% UDP. According to the description in S2, a 1.5-L reaction system was prepared by adding 113.5 g crude UTP (undried), 7.50 g kanamycin sulfate, and 3.05 g magnesium chloride hexahydrate. The pH was adjusted to 8.75 with the addition of NaOH solution. After saturation, the A262nm value was 1037. 228.76 U of pure AphIf mutant enzyme was added. The reaction was incubated in a 35°C waterbath. After 13.5 h, the residual GTP content was 0.006% and the UTP content was 92.462%.

[0120] After separation on an ion exchange column, the purity of the obtained UTP wet powder was 97.757%, and that of GTP was 0.018%; after drying, the purity of UTP was 96.425%, and that of GTP was 0.012%. Embodiment 9

[0121] Removal of GTP from UTP using aminoglycoside 2'-phosphotransferase mutants:

[0122] The crude UTP product had a UTP purity of 94.79%, with impurities of 2.43% GTP and 1.42% UDP. Following the instructions in S2, a 1.5 L reaction system was prepared by adding 93.2 g crude UTP, 3.75 g kanamycin sulfate, and 3.05 g magnesium chloride hexahydrate. The pH was adjusted to 8.75 with the addition of NaOH solution. After saturation, the A262 nm reading was 1034. 228.76 U of pure AphIf mutant enzyme was added. The reaction was incubated in a 35°C water bath. After 5 h of reaction, the residual GTP content was 0.023% and the UTP content was 94.775%.

[0123] After separation on an ion exchange column, the purity of the obtained UTP wet powder was 98.512%, and that of GTP was 0.025%; after drying, the purity of UTP was 97.234%, and that of GTP was 0.021%. Example 10

[0124] Removal of GTP from UTP using aminoglycoside 2'-phosphotransferase mutants:

[0125] The crude UTP product contained 94.56% UTP purity, 1.31% GTP impurities, and 2.42% UDP. Following the instructions in S2, prepare a 1.5 L reaction system with purified aminoglycoside 2'-phosphotransferase enzyme solution. Add 95.1 g crude UTP, 2.00 g kanamycin sulfate, and 3.05 g magnesium chloride hexahydrate. Adjust the pH to 8.75 with NaOH solution. After constant volume, the A262 nm reading was 1028. Add 114.38 U of pure AphIf mutant enzyme. Incubate the reaction in a 35°C waterbath. After 5 h, the residual GTP level was 0 and the UTP level was 94.550%.

[0126] After separation on an ion exchange column, the purity of the obtained UTP wet powder was 98.567%, and that of GTP was 0.006%; after drying, the purity of UTP was 97.458%, and that of GTP was 0.010%.

[0127] In summary, by adding the aminoglycoside 2'-phosphotransferase mutant, Examples 6 to 10 successfully removed the GTP impurity in UTP to an extremely low level. After the reaction and subsequent ion exchange column separation and purification steps, the purity of UTP was significantly improved. The purity of UTP in wet powder form was generally higher than 97%, and the purity of UTP after drying was also maintained above 95%, indicating that the aminoglycoside 2'-phosphotransferase mutant can achieve high-efficiency GTP removal and UTP purification, meeting the demand for high-purity UTP in fields such as biomedicine.

[0128] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

Claims

1. An aminoglycoside 2'-phosphotransferase mutant, characterized in that: The sequence of the aminoglycoside 2'-phosphotransferase is numbered AAW34150.1 in Genbank, the mutant is a single point mutation I220L, and the sequence of AAW34150.1 is shown in SEQ ID NO.

1.

2. The use of the aminoglycoside 2'-phosphotransferase mutant according to claim 1, characterized in that: The application is as follows: using an aminoglycoside 2'-phosphotransferase mutant to specifically degrade GTP into GDP, and then removing GDP by utilizing the adsorption difference between GDP and UTP on an anion exchange column, thereby obtaining high-purity UTP.

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