Aminoglycoside 2 '-phosphotransferase mutant and application thereof

Abstract

The invention discloses an aminoglycoside 2 '-phosphotransferase mutant and application thereof, and relates to the technical field of biotransformation, a preparation method of the aminoglycoside 2'-phosphotransferase mutant comprises the following steps: S1, cloning and expressing aminoglycoside 2 '-phosphotransferase; s2, purification of aminoglycoside 2 '-phosphotransferase; s3, determining the enzyme activity of aminoglycoside 2 '-phosphotransferase and the specificity of a substrate; s4, constructing a single point mutant; and S5, construction, expression and screening of an ApIf mutation library. The aminoglycoside 2 '-phosphotransferase mutant capable of specifically degrading GTP without degrading UTP is obtained through cloning, expression, purification and screening, and the problems that GTP is difficult to separate and purify in the UTP production process, the GTP impurity content is high in the UTP production process, and the yield and purity of UTP are low can be solved.

Description

Technical Field

[0001] The 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 GTP being difficult to separate and purify, 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 the purity of the UTP product being seriously affected. Ultimately, these problems jointly lead to the 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 GTP being difficult to separate and purify in the UTP production process, the high content of GTP impurities in the UTP production process, and the low yield and purity of UTP.

[0003] The defects of existing transferase mutants are: 1. Patent document US10519429B2 discloses a nicotinamide phosphoribosyltransferase mutant and its application. The document mainly considers the problem of how to provide a NAMPT mutant with a catalytic activity higher than that of the conventional wild-type parent, but does not consider how to solve the problem of difficulty in separating and purifying GTP in the UTP production process, high impurity content of GTP in the UTP production process, and low UTP yield and purity; 2. Patent document CN109295027B discloses a glycosyltransferase mutant. The 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; 3. Patent document CN112080480B discloses a glycosyltransferase mutant and its application. The document mainly considers how to increase the accumulation of specific steviol glycoside intermediates, but does not consider how to improve the purity of the enzyme, optimize the purification conditions, improve the activity and stability of the enzyme, achieve large-scale purification of the enzyme, and reduce the purification cost; 4. 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 prior art, 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

[0004] 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.

[0005] 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: S1, cloning and expression of aminoglycoside 2'-phosphotransferase; S2, purification of aminoglycoside 2'-phosphotransferase; S3, aminoglycoside 2'-phosphotransferase enzyme activity and substrate specificity determination; 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; S5. Construction, expression and screening of AphIf mutant library; In S2, it also includes the following: S21, suspending the wet bacterial cells obtained in S1 with buffer A, disrupting them with ultrasound, centrifuging them at 4°C and 12,000 rpm for 20 min, taking the supernatant, and obtaining a crude extract; S22. Use Ni affinity column to purify protein. The specific operation is as follows: ① Equilibrate Ni column with 5 column volumes of buffer A until the baseline is stable; ② Load crude extract sample at a flow rate of 1 mL / min, with the loading amount of 25-40 mg / mL protein, so that the target protein is adsorbed on Ni column; ③ Rinse 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, collect target protein, dialyze target protein in pH 8.0, 20 mM Tris buffer overnight to obtain purified nicotinamide phosphoribosyltransferase; ⑤ Rinse Ni column with 5 column volumes of buffer A until the baseline is stable, and preserve Ni column with 5 column volumes of ultrapure water containing 20% ​​ethanol.

[0006] Preferably, the ultrasonic crushing is carried out in an ice bath environment, the power is 400W, and the working cycle is crushing for 3 seconds and pausing for 5 seconds, which lasts for 15 minutes; Buffer A was pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 20 mM imidazole; Buffer B was pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 400 mM imidazole; The size of the Ni affinity column is 1.6×10 cm and it is produced in the United States.

[0007] Preferably, in S1, the following is also included: S11, the aminoglycoside 2'-phosphotransferase gene was optimized according to the codon preference of E. coli, the rare codons in these nucleotide sequences were added with an NcoⅠ restriction site before the start codon, the stop codon was deleted, and an XhoⅠ restriction site was added at the end of the sequence; S12, these sequences were sent to Shanghai Jierui for full gene synthesis, and connected to the pET28a(+) vector using the NcoⅠ / XhoⅠ double restriction sites to construct the pET28a(+)-AphIf expression vector, i.e., the pET28a-AphIf plasmid; S13, the constructed pET28a-AphIf plasmid was collected by centrifugation at 10000 rpm for 10 min, and 50 μL of ddH2O was added to dissolve it on a vortex shaker, and E. coli BL21 (DE3) was transformed after dissolution, and the transformed E. coli BL21 (DE3) was activated, placed at 37°C and 220 rpm, cultured for 1 hour, coated on an LB plate containing 50 μg / mL kanamycin resistance, and inverted cultured at 37°C overnight; 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, and culture at 37°C and 220rpm. When 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.

[0008] Preferably, the nucleotide sequence of the aminoglycoside 2'-phosphotransferase gene is the 9696-10589 sequence of Campylobacter jejuniplasmid pCG8245 GenBank: AY701528.1, and the amino acid sequence Genbank is AAW34150.1; The NcoⅠ restriction site is CCATGG.

[0009] Preferably, S3 further includes the following: S31. The standard conditions for enzyme activity detection are: substrate 25g / L GTP, inhibitor 25g / L kanamycin sulfate, cofactor 2.5g / LMgCl2∙6H2O, appropriate amount of enzyme solution, reaction at 35℃ and pH 8.0 for 20 minutes, sample treatment and HPLC detection analysis; Liquid phase detection conditions for S32, GDP, and GTP include the chromatographic column Inert Sustain TMC18, mobile phase was acetonitrile: 0.04M PBS = 7:93, flow rate was 1.0mL / min, detection wavelength was 262nm, injection volume was 10μL, column temperature was 40℃.

[0010] Preferably, Inert Sustain TM The size of C18 is 4.6×150mm, the particle size is 5µm, and the brand is Shimadzu; The specificity of aminoglycoside 2'-phosphotransferase was determined by replacing GTP with ATP and UTP in the standard conditions of the enzymatic assay.

[0011] Preferably, in S4, the following is also included: S41. Using homology modeling methods, 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; S42, using the flexible docking principle, in the process of molecular docking, considering the conformational changes of the ligand GTP, allowing the amino acid residues near the active site to be flexible, after removing water molecules, adding hydrogen atoms, setting pH and determining the docking box range, analyzing the binding energy and interaction force between the ligand and the protein, and selecting the most suitable docking model; S43. The protein three-dimensional structure was analyzed by PyMOL, and the "hotspot residues" were analyzed by HotSpot Wizard to construct a mutation library. The screening conditions included selecting amino acid residues that interact with GTP near GTP5Å in the most suitable docking model, and using HotSpot Wizard to automatically identify mutation hotspots. Based on the combination of the two, the "hotspot" amino acids near GTP5Å were given priority, and the active sites were excluded. Based on this, 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, Q254A.

[0012] Preferably, in S5, the following is also included: 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; S52, PCR amplification was performed using Takara's KOD high-fidelity polymerase according to amplification conditions, and then the PCR product was recovered from the gel, and the gel-recovered product was digested with DpnI enzyme to degrade the initial template, and the digested product was transformed into BL21 (DE3), and the positive clones were screened on the plate and sequenced to verify the recombinant bacteria of the AphIf enzyme mutant; 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 method to obtain mutant pure enzyme solution.

[0013] 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.

[0014] 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 difference in adsorption of GDP and UTP by an anion exchange column, thereby obtaining high-purity UTP.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention obtains an aminoglycoside 2'-phosphotransferase mutant that can specifically degrade GTP but not UTP through cloning, expression, purification and screening. UTP is often converted by whole-cell catalysis of Saccharomyces cerevisiae. However, since Saccharomyces cerevisiae is a living cell, it will carry impurity GTP. The properties of UTP and GTP are very similar, so it is difficult to separate and purify them, and the yield is extremely low. The aminoglycoside 2'-phosphotransferase mutant has a high substrate specificity, can efficiently convert GTP into GDP, and does not degrade UTP. In the production process of UTP, the enzyme mutant is introduced. The method can specifically degrade GTP, thereby reducing the content of GTP in the UTP product. At the same time, due to the difference in adsorption of GDP and UTP on the anion exchange column, GDP can be separated from UTP through the anion exchange column, thereby 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.

[0016] 2. The present invention can improve 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). The efficient expression of the enzyme is further ensured by adding appropriate restriction sites and connecting to the efficient pET28a-AphIf expression vector. At the same time, the present invention constructs the pET28a-AphIf expression vector and expresses it in E. coli The aminoglycoside 2'-phosphotransferase mutant is efficiently expressed in BL21 (DE3), which provides the possibility for the industrial production of the enzyme. By optimizing the culture conditions, a high-yield wet bacterial cell containing enzyme is obtained, which lays a foundation for the subsequent enzyme purification and application. The substrate specificity and catalytic efficiency of the enzyme mutant obtained by the method of the present invention may be optimized, thereby improving the efficiency of biotransformation and the purity of the product. 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. The antibiotic molecule is modified by phosphorylation to reduce its antibacterial activity. 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.

[0017] 3. The present invention can effectively separate the target protein, aminoglycoside 2'-phosphotransferase, from the crude extract by using Ni affinity column purification technology. After the steps of balancing with buffer A, loading the crude extract, washing with impurities and eluting with buffer B, high-purity aminoglycoside 2'-phosphotransferase is finally obtained. In the purification process, the present invention optimizes the conditions of ultrasonic crushing, centrifugation, loading, washing and elution. The ultrasonic crushing 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 crush for 3 seconds and pause for 5 seconds for 15 minutes to ensure the crushing effect while reducing energy consumption. The centrifugal conditions, loading amount, washing and elution flow rates are also aimed at improving the 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 also can be used for large-scale purification in industrial production. By adjusting the parameters such as the size of the Ni affinity column, the sample loading amount and the volume of the buffer, the production requirements 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, enzyme engineering, etc., so 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.

[0018] 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, providing a basis for subsequent molecular docking and mutant design. Through the flexible docking principle, 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 HotSpot Wizard analyzes "hot spot residues". The present invention screens out amino acid residues that interact with GTP and constructs a mutation library containing 20 single-point mutants, providing abundant candidate materials for subsequent enzyme mutant screening and performance optimization. By screening the mutation library, the present 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

[0019] Figure 1 It is a three-dimensional diagram and an active center site diagram of the aminoglycoside 2'-phosphotransferase of the present invention. DETAILED DESCRIPTION

[0020] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Embodiment 1: See also Figure 1 The present invention provides an embodiment: an aminoglycoside 2'-phosphotransferase mutant, and the preparation method of the aminoglycoside 2'-phosphotransferase mutant is as follows: S1, cloning and expression of aminoglycoside 2'-phosphotransferase; In S1, the following is also included: S11, the aminoglycoside 2'-phosphotransferase gene was optimized according to the codon preference of E. coli, the rare codons in these nucleotide sequences were added with an NcoⅠ restriction site before the start codon, the stop codon was deleted, and an XhoⅠ restriction site was added at the end of the sequence; S12, these sequences were sent to Shanghai Jierui for full gene synthesis, and connected to the pET28a(+) vector using the NcoⅠ / XhoⅠ double restriction sites to construct the pET28a(+)-AphIf expression vector, i.e., the pET28a-AphIf plasmid; S13, the constructed pET28a-AphIf plasmid was collected by centrifugation at 10000 rpm for 10 min, and 50 μL of ddH2O was added to dissolve it on a vortex shaker, and E. coli BL21 (DE3) was transformed after dissolution, and the transformed E. coli BL21 (DE3) was activated, placed at 37°C and 220 rpm, cultured for 1 hour, coated on an LB plate containing 50 μg / mL kanamycin resistance, and inverted cultured at 37°C overnight; S14. Pick a single colony from the plate and inoculate it into TB liquid medium containing a final concentration of 50 µg / mL kanamycin, add 5 g / L glucose and 20 g / L lactose, and culture at 37°C and 220 rpm. When 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.

[0022] The nucleotide sequence of the aminoglycoside 2'-phosphotransferase gene is the 9696-10589 sequence of Campylobacter jejuni plasmidpCG8245 GenBank: AY701528.1, and the amino acid sequence Genbank is AAW34150.1; The NcoⅠ restriction site is CCATGG.

[0023] Embodiment 2: An aminoglycoside 2'-phosphotransferase mutant, the preparation method of the aminoglycoside 2'-phosphotransferase mutant is as follows: S2, purification of aminoglycoside 2'-phosphotransferase; In S2, it also includes the following: S21, suspending the wet bacterial cells obtained in S1 with buffer A, disrupting them with ultrasound, centrifuging them at 4°C and 12,000 rpm for 20 min, taking the supernatant, and obtaining a crude extract; S22. Use Ni affinity column to purify protein. The specific operation is as follows: ① Equilibrate Ni column with 5 column volumes of buffer A until the baseline is stable; ② Load crude extract sample at a flow rate of 1 mL / min, with the loading amount of 25-40 mg / mL protein, so that the target protein is adsorbed on Ni column; ③ Rinse 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, collect target protein, dialyze target protein in pH 8.0, 20 mM Tris buffer overnight to obtain purified nicotinamide phosphoribosyltransferase; ⑤ Rinse Ni column with 5 column volumes of buffer A until the baseline is stable, and preserve Ni column with 5 column volumes of ultrapure water containing 20% ​​ethanol.

[0024] Ultrasonic disruption was carried out 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; Buffer A was pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 20 mM imidazole; Buffer B was pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 400 mM imidazole; The size of the Ni affinity column is 1.6×10 cm and it is produced in the United States.

[0025] Embodiment three: An aminoglycoside 2'-phosphotransferase mutant, the preparation method of the aminoglycoside 2'-phosphotransferase mutant is as follows: S3, aminoglycoside 2'-phosphotransferase enzyme activity and substrate specificity determination; In S3, it also includes the following: S31. The standard conditions for enzyme activity detection are: substrate 25g / L GTP, inhibitor 25g / L kanamycin sulfate, cofactor 2.5g / LMgCl2∙6H2O, appropriate amount of enzyme solution, reaction at 35℃ and pH 8.0 for 20 minutes, sample treatment and HPLC detection analysis; Liquid phase detection conditions for S32, GDP, and GTP include the chromatographic column Inert Sustain TM C18, mobile phase was acetonitrile: 0.04M PBS = 7:93, flow rate was 1.0mL / min, detection wavelength was 262nm, injection volume was 10μL, column temperature was 40℃.

[0026] Inert Sustain TM The size of C18 is 4.6×150mm, the particle size is 5µm, and the brand is Shimadzu; The specificity of aminoglycoside 2'-phosphotransferase was determined by replacing GTP with ATP and UTP in the standard conditions of the enzymatic assay.

[0027] Further, the measurement results are as follows:

[0028] After determination, the enzyme activity of the purified aminoglycoside 2'-phosphotransferase was 79 U / mL, the enzyme activity to ATP was 4.63 U / mL, and the enzyme activity relative to GTP was only 5.86%. The enzyme activity to UTP was 11.29 U / mL, and the enzyme activity relative to GTP was only 14.29%.

[0029] Embodiment 4: An aminoglycoside 2'-phosphotransferase mutant, the preparation method of the aminoglycoside 2'-phosphotransferase mutant is as follows: 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; In S4, it also includes the following: S41. Using homology modeling methods, 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; S42, using the flexible docking principle, in the process of molecular docking, considering the conformational changes of the ligand GTP, allowing the amino acid residues near the active site to be flexible, after removing water molecules, adding hydrogen atoms, setting pH and determining the docking box range, analyzing the binding energy and interaction force between the ligand and the protein, and selecting the most suitable docking model; S43. The protein three-dimensional structure was analyzed by PyMOL, and the "hotspot residues" were analyzed by HotSpot Wizard to construct a mutation library. The screening conditions included selecting amino acid residues that interact with GTP near GTP5Å in the most suitable docking model, and using HotSpot Wizard to automatically identify mutation hotspots. Based on the combination of the two, the "hotspot" amino acids near GTP5Å were given priority, and the active sites were excluded. Based on this, 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, Q254A.

[0030] Furthermore, the homology modeling method was used to predict the structure of AphIf protein. The amino acid sequence of AphIf was input into SWISS-MODEL, the target protein sequence was aligned with the template sequence, a preliminary three-dimensional model was constructed, and the template sequence with the highest homology to the amino acid sequence of AphIf was selected. Finally, the three-dimensional model of AphIf was obtained, which is Figure 1 .

[0031] The principle of flexible docking is adopted, and AutoDock Vina is used for docking, which allows the conformation of the ligand small molecule GTP and the amino acid residues near the active site to change during the docking process, making the predicted docking model more accurate. Water molecules are removed from the small molecule ligand and the macromolecule protein, hydrogen atoms are added, and the environmental pH is set; through the active site, the docking box range is determined, the docking file and docking script are specified, and finally the binding energy and related forces between the ligand and the protein are analyzed to select the most suitable docking model.

[0032] The three-dimensional structure of the protein was analyzed by PyMOL, and the "hotspot residues" in the protein were analyzed by HotSpot Wizard (an online tool based on semi-rational design). The mutation library was further constructed, and the screening conditions were as follows: (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.

[0033] (2) Use HotSpot Wizard to automatically identify mutation hotspots and select hotspot positions suitable for mutagenesis by integrating structural, functional and evolutionary information. These hotspots are essential for improving protein stability, catalytic activity, substrate specificity and enantioselectivity. After the protein model of AphIf is imported, 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.

[0034] (3) Combining the above two screening methods, the "hot spot" amino acids near GTP5Å are 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.

[0035] 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.

[0036] Embodiment five: An aminoglycoside 2'-phosphotransferase mutant, the preparation method of the aminoglycoside 2'-phosphotransferase mutant is as follows: S5. Construction, expression and screening of AphIf mutant library; In S5, it also includes the following: 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; S52, PCR amplification was performed using Takara's KOD high-fidelity polymerase according to amplification conditions, and then the PCR product was recovered from the gel, and the gel-recovered product was digested with DpnI enzyme to degrade the initial template, and the digested product was transformed into BL21 (DE3), and the positive clones were screened on the plate and sequenced to verify the recombinant bacteria of the AphIf enzyme mutant; 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 method to obtain mutant pure enzyme solution.

[0037] Amplification conditions were: 95oC for 2 min, then 55oC for 20 sec, 72oC for 100 sec, for a total of 30 cycles, and finally 72oC for 10 min.

[0038] Furthermore, the pET28a-AphIf plasmid in S1 was used as a template, the Quick-change mutation method was used, and the amino acids at each site were mutated using primers to construct a mutation library.

[0039]

[0040] PCR amplification used Takara's KOD high-fidelity polymerase, and the amplification conditions were: 95oC for 2min, then 55oC for 20sec, 72oC for 100sec, for a total of 30 cycles, and finally 72oC for 10min; 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 on LB agar plates containing 50 μg / mL kanamycin. The plate was cultured at 37°C overnight, positive clones were screened, and sequencing was performed to obtain recombinant bacteria with AphIf enzyme mutants.

[0041] 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:

[0042] The results showed that the activity of I220L with GTP as substrate was 20.67U / mL, while the activity with UTP as substrate was only 0.84% ​​of that with GTP as substrate, which greatly improved the substrate specificity. 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. The substrate specificity has been improved to varying degrees.

[0043] The above mutant crude enzyme solution was purified according to the method of S2 to obtain the corresponding mutant pure enzyme solution.

[0044] Embodiment six: The application of the aminoglycoside 2'-phosphotransferase mutant is as follows: GTP is specifically degraded into GDP by using the aminoglycoside 2'-phosphotransferase mutant, and GDP is then removed by using the difference in adsorption of GDP and UTP by an anion exchange column, thereby obtaining high-purity UTP.

[0045] Furthermore, aminoglycoside 2'-phosphotransferase mutants were used to remove GTP from UTP: The purity of UTP in the crude UTP product was 92.27%, the impurity GTP was 4.95%, and UDP was 1.37%. According to the description of S2, the purified aminoglycoside 2'-phosphotransferase mutant pure enzyme solution was taken, the reaction system was 1.5L, 113.5g UTP crude product (undried), 7.50g kanamycin sulfate, 3.05g magnesium chloride hexahydrate were added, and the pH was adjusted to 8.75 by adding NaOH solution. After constant volume, A262nm was measured to be 1055. 228.76U of pure enzyme of AphIf mutant was added. React in a water bath at 35℃. After 9h of reaction, the residual amount of GTP was 0.106% and that of UTP was 92.651%.

[0046] 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%.

[0047] Embodiment seven: Removal of GTP from UTP using aminoglycoside 2'-phosphotransferase mutants: The purity of UTP in crude UTP was 92.27%, the impurity GTP was 4.95%, and UDP was 1.37%. According to the description of S2, the purified aminoglycoside 2'-phosphotransferase pure enzyme solution was taken, the reaction system was 1.5L, 113.5g UTP crude (undried), 7.50g streptomycin, 3.05g magnesium chloride hexahydrate were added, and the pH was adjusted to 8.75 by adding NaOH solution. After constant volume, A262nm was measured to be 1026. 228.76U of pure enzyme of AphIf mutant was added. React in a water bath at 35℃. After 11h of reaction, the residual amount of GTP was 0.131% and that of UTP was 92.643%.

[0048] After separation on an ion exchange column and drying, the purity of UTP was 96.074% and that of GTP was 0.115%.

[0049] Embodiment eight: Removal of GTP from UTP using aminoglycoside 2'-phosphotransferase mutants: The purity of UTP in crude UTP was 92.27%, the impurity GTP was 4.95%, and UDP was 1.37%. According to the description of S2, the purified aminoglycoside 2'-phosphotransferase pure enzyme solution was taken, the reaction system was 1.5L, 113.5g UTP crude (undried), 7.50g kanamycin sulfate, 3.05g magnesium chloride hexahydrate were added, and the pH was adjusted to 8.75 by adding NaOH solution. After constant volume, A262nm was measured to be 1037. 228.76U of pure enzyme of AphIf mutant was added. React in a water bath at 35℃. After 13.5h of reaction, the residual amount of GTP was 0.006%, and that of UTP was 92.462%.

[0050] 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%.

[0051] Embodiment nine: Removal of GTP from UTP using aminoglycoside 2'-phosphotransferase mutants: The purity of UTP in crude UTP was 94.79%, the impurity GTP was 2.43%, and UDP was 1.42%. According to the description of S2, the purified aminoglycoside 2'-phosphotransferase mutant pure enzyme solution was taken, the reaction system was 1.5L, 93.2g UTP crude, 3.75g kanamycin sulfate, 3.05g magnesium chloride hexahydrate were added, and NaOH solution was added to adjust the pH to 8.75. After constant volume, A262nm was measured to be 1034. 228.76U of pure enzyme of AphIf mutant was added. React in a 35℃ water bath. After 5h of reaction, the residual GTP was 0.023% and UTP was 94.775%.

[0052] 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%.

[0053] Embodiment ten: Removal of GTP from UTP using aminoglycoside 2'-phosphotransferase mutants: The purity of UTP in crude UTP was 94.56%, the impurity GTP was 1.31%, and UDP was 2.42%. According to the description of S2, the purified aminoglycoside 2'-phosphotransferase pure enzyme solution was taken, the reaction system was 1.5L, 95.1g UTP crude, 2.00g kanamycin sulfate, 3.05g magnesium chloride hexahydrate were added, and the pH was adjusted to 8.75 by adding NaOH solution. After constant volume, A262nm was measured to be 1028. 114.38U of pure enzyme of AphIf mutant was added. React in a water bath at 35℃. After 5h of reaction, the residual GTP was 0 and the UTP was 94.550%.

[0054] 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%.

[0055] 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 efficient GTP removal and UTP purification, meeting the demand for high-purity UTP in the fields of biomedicine and the like.

[0056] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. An aminoglycoside 2'-phosphotransferase mutant, characterized in that: The preparation method of aminoglycoside 2'-phosphotransferase mutant is as follows: S1, cloning and expression of aminoglycoside 2'-phosphotransferase; S2, purification of aminoglycoside 2'-phosphotransferase; S3, aminoglycoside 2'-phosphotransferase enzyme activity and substrate specificity determination; 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; S5. Construction, expression and screening of AphIf mutant library; In S2, it also includes the following: S21, suspending the wet bacterial cells obtained in S1 with buffer A, disrupting them with ultrasound, centrifuging them at 4°C and 12,000 rpm for 20 min, taking the supernatant, and obtaining a crude extract; S22. Use Ni affinity column to purify protein. The specific operation is as follows: ① Equilibrate Ni column with 5 column volumes of buffer A until the baseline is stable; ② Load crude extract sample at a flow rate of 1 mL / min, with the loading amount of 25-40 mg / mL protein, so that the target protein is adsorbed on Ni column; ③ Rinse 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, collect target protein, dialyze target protein in pH 8.0, 20 mM Tris buffer overnight to obtain purified nicotinamide phosphoribosyltransferase; ⑤ Rinse Ni column with 5 column volumes of buffer A until the baseline is stable, and preserve Ni column with 5 column volumes of ultrapure water containing 20% ​​ethanol.

2. An aminoglycoside 2'-phosphotransferase mutant according to claim 1, characterized in that: Ultrasonic disruption was carried out 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; Buffer A was pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 20 mM imidazole; Buffer B was pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 400 mM imidazole; The size of the Ni affinity column is 1.6×10 cm and it is produced in the United States.

3. An aminoglycoside 2'-phosphotransferase mutant according to claim 1, characterized in that: In S1, the following are also included: S11, the aminoglycoside 2'-phosphotransferase gene was optimized according to the codon preference of E. coli, the rare codons in these nucleotide sequences were added with an NcoⅠ restriction site before the start codon, the stop codon was deleted, and an XhoⅠ restriction site was added at the end of the sequence; S12, these sequences were sent to Shanghai Jierui for full gene synthesis, and connected to the pET28a(+) vector using the NcoⅠ / XhoⅠ double restriction sites to construct the pET28a(+)-AphIf expression vector, i.e., the pET28a-AphIf plasmid; S13, the constructed pET28a-AphIf plasmid was collected by centrifugation at 10000 rpm for 10 min, and 50 μL of ddH2O was added to dissolve it on a vortex shaker, and E. coli BL21 (DE3) was transformed after dissolution, and the transformed E. coli BL21 (DE3) was activated, placed at 37°C and 220 rpm, cultured for 1 hour, coated on an LB plate containing 50 μg / mL kanamycin resistance, and inverted cultured at 37°C overnight; S14. Pick a single colony from the plate and inoculate it into TB liquid medium containing a final concentration of 50 µg / mL kanamycin, add 5 g / L glucose and 20 g / L lactose, and culture at 37°C and 220 rpm. When 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.

4. An aminoglycoside 2'-phosphotransferase mutant according to claim 3, characterized in that: The nucleotide sequence of the aminoglycoside 2'-phosphotransferase gene is the 9696-10589 sequence of Campylobacter jejuni plasmid pCG8245 GenBank: AY701528.1, and the amino acid sequence Genbank is AAW34150.1; The NcoⅠ restriction site is CCATGG.

5. The aminoglycoside 2'-phosphotransferase mutant according to claim 1, characterized in that: In S3, it also includes the following: S31. The standard conditions for enzyme activity detection are: substrate 25g / L GTP, inhibitor 25g / L kanamycin sulfate, cofactor 2.5g / LMgCl2∙6H2O, appropriate amount of enzyme solution, reaction at 35℃ and pH 8.0 for 20 minutes, sample treatment and HPLC detection analysis; Liquid phase detection conditions for S32, GDP, and GTP include the chromatographic column Inert Sustain TM C18, mobile phase was acetonitrile:0.04MPBS=7:93, flow rate was 1.0mL / min, detection wavelength was 262nm, injection volume was 10μL, column temperature was 40℃.

6. An aminoglycoside 2'-phosphotransferase mutant according to claim 5, characterized in that: InertSustain TM The size of C18 is 4.6×150mm, the particle size is 5µm, and the brand is Shimadzu; The specificity of aminoglycoside 2'-phosphotransferase was determined by replacing GTP with ATP and UTP in the standard conditions of the enzymatic assay.

7. The aminoglycoside 2'-phosphotransferase mutant according to claim 1, characterized in that: In S4, it also includes the following: S41. Using homology modeling methods, 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; S42, using the flexible docking principle, in the process of molecular docking, considering the conformational changes of the ligand GTP, allowing the amino acid residues near the active site to be flexible, after removing water molecules, adding hydrogen atoms, setting pH and determining the docking box range, analyzing the binding energy and interaction force between the ligand and the protein, and selecting the most suitable docking model; S43. The protein three-dimensional structure was analyzed by PyMOL, and the "hotspot residues" were analyzed by HotSpot Wizard to construct a mutation library. The screening conditions included selecting amino acid residues that interact with GTP near GTP5Å in the most suitable docking model, and using HotSpot Wizard to automatically identify mutation hotspots. Based on the combination of the two, the "hotspot" amino acids near GTP5Å were given priority, and the active sites were excluded. Based on this, 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, Q254A.

8. The aminoglycoside 2'-phosphotransferase mutant according to claim 1, characterized in that: In S5, the following are also included: 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; S52, PCR amplification was performed using Takara's KOD high-fidelity polymerase according to amplification conditions, and then the PCR product was recovered from the gel, and the gel-recovered product was digested with DpnI enzyme to degrade the initial template, and the digested product was transformed into BL21 (DE3), and the positive clones were screened on the plate and sequenced to verify the recombinant bacteria of the AphIf enzyme mutant; 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 method to obtain mutant pure enzyme solution.

9. An aminoglycoside 2'-phosphotransferase mutant according to claim 8, characterized in that: Amplification conditions were: 95oC for 2 min, then 55oC for 20 sec, 72oC for 100 sec, for a total of 30 cycles, and finally 72oC for 10 min.

10. Use of an aminoglycoside 2'-phosphotransferase mutant, applicable to an aminoglycoside 2'-phosphotransferase mutant according to any one of claims 1 to 9, characterized in that: The application of the aminoglycoside 2'-phosphotransferase mutant is as follows: GTP is specifically degraded into GDP by using the aminoglycoside 2'-phosphotransferase mutant, and GDP is then removed by using the difference in adsorption of GDP and UTP by an anion exchange column, thereby obtaining high-purity UTP.

Images