5'-Methylthioadenosine phosphorylase mutant and its application

Abstract

The present invention provides a 5'-methylthioadenosine phosphorylase mutant, the amino acid sequence of which is derived from a mutation of the sequence shown in SEQ ID No. 3 of the sequence table, wherein the mutation is selected from at least one of the following: H64A, P131D, W114N, L206S, E168Q, F217V, F217S, F217W, R219A, R219Q, R219F, V184A and V184T. The present invention also provides its application in the catalytic synthesis of 2'-fluoro-2'-deoxyadenosine. The mutant H64A / W114N / L206S of the present invention has an enzyme activity of 1.31 U / g, while the ApMTAP unmutated enzyme strain has an enzyme activity of 0.11 U / g, which is about 12 times higher than the enzyme activity, that is, the yield of 2'-fluoro-2'-deoxyadenosine has been greatly improved, and is suitable for the field of enzyme catalysis.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, relates to a 5'-methylthioadenosine phosphorylase mutant and its application, and particularly relates to its application in catalyzing the synthesis of 2'-fluoro-2'-deoxyadenosine. Background Art

[0002] Nucleosides and their analogs have long been used as small molecule drugs and are widely used in cancer and antiviral treatments, including but not limited to valacyclovir for the treatment of HSV-1 and cytarabine for the treatment of specific leukemias. Interest in nucleoside analogs has greatly increased, and multiple nucleoside analogs or combinations have been approved for the treatment of various human viral infections, including HSV, varicella-zoster virus (VZV), hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), and human cytomegalovirus (HCMV).

[0003] In particular, pyrimidine and purine nucleosides modified at the 2'-carbon atom of the pentose ring are of great medicinal significance. Substitution of hydrogen or hydroxyl groups in the pentose moiety of nucleosides with fluorine has yielded analogs with diverse biological activities. 2'-Fluorinated nucleosides have been shown to possess favorable properties as components of antisense oligonucleotides and small interfering RNAs, exhibiting superior therapeutic efficacy compared to other C2'-modified nucleotides in disease treatment. Nucleoside analogs with 2'-deoxy-2'-fluororibosyl moieties have been studied and have demonstrated activity against herpes simplex virus, pseudorabies virus, equine abortion virus, influenza virus, and varicella virus, with some also inhibiting the growth of leukemia cells.

[0004] The importance of nucleoside analogs has driven researchers to pursue greener, more efficient, and simpler synthetic methods. Enzyme catalysis has attracted attention due to its advantages, such as higher regio- and stereoselectivity and higher product purity. However, the lack of enzymes with broad substrate specificity limits the scope of application of this strategy. Furthermore, when operated at high temperatures, the efficiency of this process is often reduced due to the thermal instability of the biocatalyst. Therefore, the search for enzymes with improved properties is of great significance in this research field. Among them, nucleoside phosphorylase catalyzes the reversible cleavage of the N-glycosidic bond of nucleosides. Therefore, under the catalytic action of nucleoside phosphorylase, the condensation of nucleobases and pentose 1-phosphate can be used to synthesize nucleosides or their analogs. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention provides mutants of 5'-methylthioadenosine phosphorylase and their applications, specifically their applications in the catalytic synthesis of 2'-fluoro-2'-deoxyadenosine. The present invention molecularly modifies wild-type 5'-methylthioadenosine phosphorylase ApMTAP through site-directed mutagenesis, addressing its lack of substrate specificity and instability. This dual-enzyme cascade catalyzes the synthesis of 2'-fluoro-2'-deoxyadenosine with the pyrimidine nucleoside phosphorylase TtPyNP, enhancing enzyme activity and increasing the yield of 2'-fluoro-2'-deoxyadenosine.

[0006] The first object of the present invention is to provide a 5'-methylthioadenosine phosphorylase mutant, the amino acid sequence of which is derived by mutating the sequence shown in SEQ ID No. 3 in the sequence listing, wherein the mutation is selected from at least one of the following: H64A, P131D, W114N, L206S, E168Q, F217V, F217S, F217W, R219A, R219Q, R219F, V184A and V184T.

[0007] Preferably, the mutation is selected from at least two of the following: H64A, P131D, W114N and L206S;

[0008] Preferably, the mutation is: H64A+W114N+L206S.

[0009] The second object of the present invention is to provide a nucleotide sequence encoding the above-mentioned 5'-methylthioadenosine phosphorylase mutant.

[0010] Preferably, the nucleotide sequence is SEQ ID NO.6.

[0011] The third object of the present invention is to provide a recombinant expression vector comprising the above nucleotide sequence;

[0012] Preferably, the recombinant expression vector comprises pET-28a(+).

[0013] The fourth object of the present invention is to provide a genetically engineered bacterium comprising the above-mentioned recombinant expression vector;

[0014] Preferably, the genetically engineered bacteria is a recombinant strain obtained by connecting the above nucleotide sequence to a vector to obtain a recombinant expression vector, which is then introduced into a host bacterium for induction of expression, wherein the host bacterium includes Escherichia coli, Bacillus subtilis or yeast;

[0015] Preferably, the Escherichia coli is the Escherichia coli BL21 (DE3) strain.

[0016] The fifth object of the present invention is to provide the use of the 5'-methylthioadenosine phosphorylase mutant, nucleotide sequence, recombinant expression vector, and genetically engineered bacteria in catalyzing the synthesis of 2'-fluoro-2'-deoxyadenosine.

[0017] Preferably, the application is specifically as follows: adding 2'-fluoro-2'-deoxyuridine, adenine, pyrimidine nucleoside phosphorylase TtPyNP and the above-mentioned 5'-methylthioadenosine phosphorylase mutant into the reaction system, and reacting at 50-90°C;

[0018] Preferably, a crude enzyme solution of the M8 mutant and TtPyNP is prepared using a phosphate buffer having a pH of 5.5-8.5 and a concentration of 8-50 mM; more preferably, a crude enzyme solution of the M8 mutant and TtPyNP is prepared using a phosphate buffer having a pH of 6.0 and a concentration of 10 mM;

[0019] Preferably, the molar ratio of 2'-fluoro-2'-deoxyuridine to adenine is 1:0.5-1:5; more preferably, the molar ratio of 2'-fluoro-2'-deoxyuridine to adenine is 1:2;

[0020] As further preferred, the amount of 2'-fluoro-2'-deoxyuridine added is 5-50 mM; preferably, the amount of 2'-fluoro-2'-deoxyuridine added is 30-50 mM;

[0021] Preferably, the protein weight ratio of the pyrimidine nucleoside phosphorylase TtPyNP and the 5'-methylthioadenosine phosphorylase mutant is 1:0.5-1:5; further preferably, the protein weight ratio of the pyrimidine nucleoside phosphorylase TtPyNP and the 5'-methylthioadenosine phosphorylase mutant is 1:3;

[0022] Preferably, in the reaction system, the protein content of the pyrimidine nucleoside phosphorylase TtPyNP is 1-3 mg / mL, and the protein content of the 5'-methylthioadenosine phosphorylase mutant is 3-9 mg / mL; further preferably, in the reaction system, the protein content of the pyrimidine nucleoside phosphorylase TtPyNP is 2.5 mg / mL, and the protein content of the 5'-methylthioadenosine phosphorylase mutant is 7.5 mg / mL;

[0023] Preferably, the reaction time is 1-96h, more preferably the reaction time is 24-72h;

[0024] Preferably, the reaction temperature is 70°C.

[0025] A sixth object of the present invention is to provide a method for producing 2'-fluoro-2'-deoxyadenosine, comprising adding 2'-fluoro-2'-deoxyuridine, adenine, pyrimidine nucleoside phosphorylase TtPyNP, and the aforementioned 5'-methylthioadenosine phosphorylase mutant to a reaction system, and conducting the reaction at 50-90°C.

[0026] Preferably, a crude enzyme solution of the M8 mutant and TtPyNP is prepared using a phosphate buffer having a pH of 5.5-8.5 and a concentration of 8-50 mM; more preferably, a crude enzyme solution of the M8 mutant and TtPyNP is prepared using a phosphate buffer having a pH of 6.0 and a concentration of 10 mM;

[0027] Preferably, the molar ratio of 2'-fluoro-2'-deoxyuridine to adenine is 1:0.5-1:5; more preferably, the molar ratio of 2'-fluoro-2'-deoxyuridine to adenine is 1:2;

[0028] As further preferred, the amount of 2'-fluoro-2'-deoxyuridine added is 5-50 mM; preferably, the amount of 2'-fluoro-2'-deoxyuridine added is 30-50 mM;

[0029] Preferably, the protein weight ratio of the pyrimidine nucleoside phosphorylase TtPyNP and the 5'-methylthioadenosine phosphorylase mutant is 1:0.5-1:5; further preferably, the protein weight ratio of the pyrimidine nucleoside phosphorylase TtPyNP and the 5'-methylthioadenosine phosphorylase mutant is 1:3;

[0030] Preferably, in the reaction system, the protein content of the pyrimidine nucleoside phosphorylase TtPyNP is 1-3 mg / mL, and the protein content of the 5'-methylthioadenosine phosphorylase mutant is 3-9 mg / mL; further preferably, in the reaction system, the protein content of the pyrimidine nucleoside phosphorylase TtPyNP is 2.5 mg / mL, and the protein content of the 5'-methylthioadenosine phosphorylase mutant is 7.5 mg / mL;

[0031] Preferably, the reaction time is 1-96h, more preferably the reaction time is 24-72h;

[0032] Preferably, the reaction temperature is 70°C.

[0033] The present invention utilizes pyrimidine nucleoside phosphorylase to catalyze the cleavage of 2'-fluoro-2'-deoxyuridine into phosphorylated 2'-fluoropentose and free uracil, and then utilizes 5'-methylthioadenosine phosphorylase to catalyze the combination of the intermediate product phosphorylated 2'-fluoropentose and adenine to prepare 2'-fluoro-2'-deoxyadenosine.

[0034] The present invention utilizes semi-rational design to improve the catalytic performance of 5'-methylthioadenosine phosphorylase (ApMTAP) derived from Aeropyrumpernix K1. The optimal triple mutant H64A / W114N / L206S screened out has a specific enzyme activity of 1.31 U / g, while the ApMTAP unmutated enzyme strain has an activity of 0.11 U / g. The specific enzyme activity is increased by approximately 12 times, that is, the yield of 2'-fluoro-2'-deoxyadenosine is greatly improved, and the enzyme is suitable for the field of enzyme catalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0036] Figure 1 Liquid phase diagram of the production of 2'-fluoro-2'-deoxyadenosine using 2'-fluoro-2'-deoxyuridine and adenine as substrates.

[0037] Figure 2 Modeling of the 5'-methylthioadenosine phosphorylase (ApMTAP) protein structure using AlphaFold 2.

[0038] Figure 3 The enzymatic activity of ApMTAP unmutated enzyme and M8 mutant at 70℃ was measured. DETAILED DESCRIPTION

[0039] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies. The quantitative tests in the following examples were performed in triplicate, and the results were averaged.

[0040] Example 1 Construction of TtPyNP plasmid

[0041] The protein sequence of pyrimidine nucleoside phosphorylase from Thermus thermophilus was obtained from NCBI, as shown in SEQ ID NO. 1. After codon optimization, the DNA sequence encoding the enzyme was obtained, as shown in SEQ ID NO. 2. This DNA sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd. and cloned into the corresponding vector to prepare TtPyNP plasmid powder.

[0042] Protein sequence of pyrimidine nucleoside phosphorylase (SEQ ID NO.1):

[0043] MNPVAFIREKREGKKHRREDLEAFLLGYLRDEVPDYQVSAWLMAAFLRGLDPEETLWLTETMARSGKVLDLSGLPHPVDKHSTGGVGDKVSLVVGPILAASGCTFAKMSGRGLAHTGGTIDKLESVPGWRGEMTEAEFLERARRVGLVIAAQSPDLAPLDGKLYALRDVTATVESVPLIASSIMSKKLAAGARSIVLDVKVGRGAFMKTLEEARLLAKTMVAIGQGAGRRVRALLTSMEAPLGRAVGNAIEVREAIEALKGEGPGDLLEVALALAEEALRLEGLDPALARKALEGGAALEKFRAFLEAQGGDPRAVEDFSLLPLAEEHPLRAEREGVVREVDAYKVGLAVLALGGGRKRKGEPIDHGVGVYLLKKPGDRVERGEALALVYHRRRGLEEALGHLREAYALGEEAHPAPLVLEAI

[0044] Codon-optimized DNA sequence of pyrimidine nucleoside phosphorylase (SEQ ID NO.2):

[0045]

[0046] Example 2 Construction of ApMTAP plasmid

[0047] The protein sequence of 5'-methylthioadenosine phosphorylase from Aeropyrumpernix K1 was obtained from NCBI, as shown in SEQ ID NO. 3. After codon optimization, the DNA sequence encoding the enzyme was obtained, as shown in SEQ ID NO. 4. This was synthesized by Beijing Qingke Biotechnology Co., Ltd. and cloned into the corresponding vector to prepare ApMTAP plasmid powder.

[0048] Protein sequence of 5'-methylthioadenosine phosphorylase (SEQ ID NO. 3):

[0049] MRKPVHLEAGPGDVAPLVVAVGDPGRAERLATGLLEDARLVSSARGLKVYTGSFNGSEVTIATHGIGGPSAAVVFEELRMLGAEVLVRLGTSGGLSKDLRLGDVVVAAGAGCYWGSGGSIQY AGERPMCLPASPDPILTAGIYRGLSSRLGDRVVLAPVMSSDAFYAETPEAAGRWRSLLGMAAVEMELHTLFSISWIRGFRSAGVLIVSDLLLPEGFKRITPGELARREVEVGRALLEVLTGGV

[0050] The codon-optimized DNA sequence of 5'-methylthioadenosine phosphorylase (SEQ ID NO. 4):

[0051] ATGCGTAAACCGGTTCATCTGGAAGCAGGTCCGGGTGATGTTGCCCCTCTGGTTGTTGCCGTTGGCGATCCTGGTCGTGCCGAACGTCTGGCAACCGGTCTGCTGGAAGATGCACGTCTGGTTAGCTCTGCACGTGGTCTGAAAGTTTATACGGGTAGTTTTAATGGTAGTGAAGTTACCATTGCCACTCATGGTATTGGTGGTCCTTCTGCAGCAGTTGTTTTCGAAGAACTGCGTATGCTGGGTGCAGAAGTTCTGGTGCGTCTGGGTACAAGCGGTGGTCTGAGCAAAGATCTGCGCCTGGGCGATGTTGTTGTTGCGGCTGGTGCAGGTTGTTATTGGGGTAGTGGTGGTAGTATTCAGTATGCAGGTGAACGTCCTATGTGTCTGCCGGCGAGTCCTGATCCGATTCTGACCGCTGGTATTTATCGTGGTCTGAGTAGCCGTCTGGGTGATCGTGTTGTTCTGGCACCGGTTATGAGCAGTGATGCATTTTATGCAGAAACCCCTGAAGCAGCCGGTCGTTGGCGTTCACTGGGTATGGCTGCCGTTGAAATGGAACTGCATACGCTGTTTTCTATTAGCTGGATTCGTGGTTTTCGTAGCGCAGGTGTTCTGATTGTTTCTGATCTGCTGCTGCCGGAAGGTTTTAAACGTATTACCCCTGGTGAACTGGCACGTCGTGAAGTTGAAGTTGGTCGTGCGCTGCTGGAAGTTCTGACCGGTGGTGTT

[0052] Example 3 Plasmid Transformation

[0053] The pyrimidine nucleoside phosphorylase plasmid powder (4 μg) obtained in Example 1 or the 5'-methylthioadenosine phosphorylase plasmid powder (4 μg) obtained in Example 2 was added to 100 μL of sterile water and shaken to mix thoroughly. 5 μL was added to 50 μL of E. coli Bl21 (DE3) competent cells and flicked to mix thoroughly. The cells were placed on ice for 30 minutes, heat-shocked in a 42°C water bath for 45 seconds, and ice-bathed for 2 minutes before adding 500 μL of sterile, non-resistant LB liquid medium. After mixing thoroughly, the cells were placed on a shaker at 37°C for activation for 1 hour. The supernatant was then centrifuged at 10,000 rpm for 1 minute, 300 μL of the supernatant was removed, the remaining liquid was mixed with the bacterial pellet, and 50 μL was spread onto a plate containing kanamycin on a solid LB medium plate. The cells were then incubated in a 37°C incubator for 16 hours.

[0054] Example 4 Enzyme Solution Preparation

[0055] Dual-enzyme fermentation: A single colony was selected from the plate obtained in Example 3 and transferred to 10 mL of LB liquid medium containing kanamycin. After incubation at 37°C for 16 h, the colony was inoculated into a conical flask containing 50 mL of TB liquid medium at a 1% inoculum size and incubated at 37°C for 16 h. Expression was induced by adding IPTG to a final concentration of 0.5 mM and inducing at 20°C for 18 h. The resulting culture was centrifuged at 8000 rpm for 5 min, and the pellet was collected and stored at -20°C until further use.

[0056] Preparation of enzyme solution: Resuspend the bacterial pellet in 50 mL of PBS and, after homogenization, disrupt the cells using a cell disruptor at a pressure between 700 and 900 bar for 45 seconds. Centrifuge the disrupted bacterial suspension at 12,000 rpm for 15 minutes, collect the supernatant, and remove any remaining cell debris.

[0057] Example 5 Enzyme activity determination

[0058] To a 2 mL reaction system, add 0.00049 g of 2'-fluoro-2'-deoxyuridine and 0.00054 g of adenine. Add only 1 mL of crude TtPyNP enzyme solution in 10 mM PBS, pH 6.0, and react for 4 hours at 70°C to allow sufficient phosphorylated 2'-fluoropentose intermediates. Then, add 1 mL of crude 5'-methylthioadenosine phosphorylase enzyme solution and react for 1 hour. The reaction solution is filtered, and the yield of 2'-fluoro-2'-deoxyadenosine is determined by high-performance liquid chromatography. Enzyme activity is defined as 1 μM of 2'-fluoro-2'-deoxyadenosine generated in 1 minute (1 U).

[0059] Example 6 High Performance Liquid Chromatography Detection Method

[0060] A C18 column was used, the mobile phase was 90% water and 10% acetonitrile (volume ratio), the flow rate was 1 mL / min, the sample load was 10 μL, and the detection wavelength was 260 nm. The peak time of uracil was about 3.5 min, the peak time of adenine was about 4.3 min, the peak time of 2'-fluoro-2'-deoxyuridine was about 4.6 min, and the peak time of 2'-fluoro-2'-deoxyadenosine was about 8.0 min. The experimental results are shown in Figure 1 .

[0061] Figure 1 Liquid phase diagram of the production of 2'-fluoro-2'-deoxyadenosine using 2'-fluoro-2'-deoxyuridine and adenine as substrates.

[0062] Example 7 Primary Screening of 5'-Methylthioadenosine Phosphorylase Mutants

[0063] The present invention uses AlphaFold 2 to perform structural modeling on the 5'-methylthioadenosine phosphorylase (abbreviated as "ApMTAP unmutated enzyme") constructed according to the methods of Examples 3 and 4 of the present invention using the ApMTAP plasmid constructed in Example 2 as a template, and performs codon optimization again. It is molecularly docked with the substrate adenine and the intermediate product 1'-phospho-2'-fluoropentose on pymol. After the docking is completed, the surrounding amino acids and other amino acids that may affect the catalytic performance are site-directed mutagenesis. The initial screening results of the 5'-methylthioadenosine phosphorylase mutants are shown in Table 1, and primers are designed using SnapGene.

[0064] Figure 2 Modeling of the 5'-methylthioadenosine phosphorylase (ApMTAP) protein structure using AlphaFold 2.

[0065] PCR amplification was performed using the ApMTAP unmutated enzyme plasmid as a template, using the designed primers and KOD DNA polymerase. After verification by agarose gel electrophoresis, the PCR product was added with the DpnI restriction enzyme to remove the template. The final reaction product was then transformed into Escherichia coli BL21(DE3) according to the method in Example 3. Crude mutant enzyme solutions were obtained using the method in Example 4. The relative activities were measured using liquid chromatography, and the four optimal single-point mutants were screened: H64A, W114N, P131D, and L206S.

[0066] It can also be handed over to a biological company to directly synthesize the gene sequences containing the site-directed mutation sites.

[0067] Table 1 Preliminary screening of 5'-methylthioadenosine phosphorylase mutants

[0068]

[0069]

[0070]

[0071] The letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

[0072] Table 2 Primer design for some mutants

[0073] Primer name Primer sequence: 5'-3' H64A-F CATTGCCACTGCTGGTATTGGTGGTCCTTCTGCAGC H64A-R GACCACCAATACCAGCAGTGGCAATGGTAACTTCACTACC W114N-F GGTGCAGGTTGTTATAATGGTAGTGGGTGGTAGTATTCAGTATGCAG W114N-R CCACCACTACCATTATAACAACCTGCACCAGCCGCAAC P131D-F GTCCTATGTGTCTGGATGCGAGTCCTGATCCGATTCTGAC P131D-R GATCAGGACTCGCATCCAGACACATAGGACGTTCACCTG L206S-F GCGCAGGTGTTTCTATTGTTTCTG L206S-R GATCAGAAACAATAGAAACACCTGCG

[0074] The ApMTAP unmutated enzyme plasmid was used as a template and site-directed mutagenesis primers were used for site-directed mutagenesis. The site-directed mutagenesis PCR reaction system is shown in Table 3, and the PCR reaction procedure is shown in Table 4.

[0075] Table 3 PCR reaction system

[0076] PCR reaction system Volume / μL Sterile water 38 KODOne™ PCR Master Mix 50 plasmids 6 Upstream primer 3 Downstream primer 3

[0077] Table 4 PCR reaction program

[0078]

[0079] Example 8 Rescreening of 5'-Methylthioadenosine Phosphorylase Mutants

[0080] The four most active single mutants were screened and combined, specifically: H64A+W114N, H64A+P131D, H64A+L206S, W114N+P131D, W114N+L206S, P131D+L206S, H64A+W114N+P131D, H64A+W114N+L206S, H64A+P131D+L206S, W114N+P131D+L206S, or H64A+W114N+P131D+L206S. The enzyme activities were measured as described in Example 5, and the results are shown in Table 5.

[0081] Table 5 Rescreening of 5'-methylthioadenosine phosphorylase mutants

[0082] name mutation site Specific enzyme activity (U / g) ApMTAP none 0.11 M1 H64A / P131D 0.74 M2 H64A / L206S 1.08 M3 W114N / P131D 0.69 M4 H64A / W114N 0.92 M5 P131D / L206S 0.86 M6 W114N / L206S 0.97 M7 H64A / W114N / P131D 0.53 M8 H64A / W114N / L206S 1.31 M9 W114N / P131D / L206S 1.17 M10 H64A / P131D / L206S 1.09 M11 H64A / W114N / P131D / L206S 0.26

[0083] As shown in Table 5, the best mutant is M8.

[0084] Amino acid sequence of 5'-methylthioadenosine phosphorylase mutant M8 (SEQ ID NO.5):

[0085] MRKPVHLEAGPGDVAPLVVAVGDPGRAERLATGLLEDARLVSSARGLKVYTGSFNGSEVTIATAGIGGPSAAVVFEELRMLGAEVLVRLGTSGGLSKDLRLGDVVVAAGAGCYNGSGGSIQY AGERPMCLPASPDPILTAGIYRGLSSRLGDRVVLAPVMSSDAFYAETPEAAGRWRSLLGMAAVEMELHTLFSISWIRGFRSAGVSIVSDLLLPEGFKRITPGELARREVEVGRALLEVLTGGV

[0086] Nucleotide sequence of 5'-methylthioadenosine phosphorylase mutant M8 (SEQ ID NO.6):

[0087] ATGCGTAAACCGGTTCACCTGGAAGCGGGGCCGGGTGATGTTGCGCCGCTGGTTGTTGCGGTTGGCGATCCGGGTCGTGCGGAACGTCTGGCGACCGGTCTGCTGGAAGATGCGCGTCTGGTTTCGAGCGCGCGTGGTCTGAAAGTTTATACCGGCTCTTTCAACGGTAGCGAAGTTACCATC GCGACCGCGGGTATCGGCGGCCCGTCTGCTGCCGTGGTTTCGAAGAACTCCGTATGCTGGGCGCGGAAGTTCTGGTTCGCCTGGGCACCAGTGGAGGCCTGTCTAAAGATTTACGTTTAGGTGATGTAGTTGTTGCTGCAGGTGCGGGTTGTTATAACGGCTCCGGTGGTTCAATTCAGTAT GCTGGTGAACGCCCGATGTGCTTACCGGCTTCTCCGGATCCGATTCTGACTGCTGGAATTTACCGTGGTCTTTCCAGCCGTTTAGGTGACCGTGTAGTTTTAGCTCCAGTTATGTCCTCTGATGCTTTTTACGCTGAAACCCCGGAAGCGGCTGGTCGCTGGCGCTCTTTAGGTATGGCGGCA GTTGAAATGGAACTGCACACCCTGTTTAGCATTTCTTGGATCCGTGGTTTCCGTAGTGCGGGTGTTTCTATTGTTTCTGATTTACTGCTGCCGGAAGGTTTTAAACGTATCACCCCAGGTGAACTGGCTCGTCGTGAAGTTGAAGTTGGTCGTGCTTTACTGGAAGTTCTGACTGGTGGTGTT

[0088] Example 9 Reaction Temperature Optimization

[0089] TtPyNP and the optimal mutant M8 obtained in Example 8 were processed according to the steps in Example 4, wherein the concentration of phosphate buffered saline (PBS) was 10 mM and the pH was 7.0, to prepare crude enzyme solutions of TtPyNP and the M8 mutant.

[0090] In a 2 mL reaction system, 0.0025 g of 2'-fluoro-2'-deoxyuridine and 0.0027 g of adenine, 1 mL of TtPyNP crude enzyme solution (protein concentration of 2 mg / mL) and 1 mL of M8 mutant crude enzyme solution (protein concentration of 2 mg / mL) were added, and the reaction was carried out at different temperatures of 50°C, 60°C, 70°C, 80°C, and 90°C for 24 h. The yield of 2'-fluoro-2'-deoxyadenosine was measured using high performance liquid chromatography according to the method described in Example 6. The yields of 2'-fluoro-2'-deoxyadenosine obtained under different reaction temperature conditions are shown in Table 6.

[0091] Table 6 Reaction temperature optimization

[0092]

[0093]

[0094] Example 10 Phosphate Buffer pH Optimization

[0095] Phosphate-buffered saline (PBS) with a pH of 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 and a concentration of 10 mM was prepared, and TtPyNP and the optimal mutant M8 obtained in Example 8 were respectively operated according to the steps in Example 4 to prepare crude enzyme solutions of TtPyNP and M8 mutant at different pH values.

[0096] To a 2 mL reaction system, 0.0025 g of 2'-fluoro-2'-deoxyuridine and 0.0027 g of adenine were added, along with 1 mL of crude TtPyNP enzyme solution (protein concentration: 2 mg / mL) and 1 mL of crude M8 mutant enzyme solution (protein concentration: 2 mg / mL). The reaction was incubated at 70°C for 24 h. The yield of 2'-fluoro-2'-deoxyadenosine was determined by high-performance liquid chromatography (HPLC) as described in Example 6. The yields of 2'-fluoro-2'-deoxyadenosine obtained under phosphate buffer conditions at different pH values ​​are shown in Table 7.

[0097] Table 7 Phosphate buffer pH optimization

[0098] pH Yield / % 5.5 32.16 6.0 34.98 6.5 33.75 7.0 31.60 7.5 26.82 8.0 19.78 8.5 18.62

[0099] Example 11 Optimization of phosphate buffer concentration

[0100] Phosphate-buffered saline (PBS) with a pH of 6.0 and concentrations of 8 mM, 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM was prepared. TtPyNP and the optimal mutant M8 obtained in Example 8 were respectively subjected to the steps in Example 4 to prepare crude enzyme solutions of TtPyNP and the M8 mutant at different concentrations.

[0101] To a 2 mL reaction system, 0.0025 g of 2'-fluoro-2'-deoxyuridine, 0.0027 g of adenine, 1 mL of crude TtPyNP enzyme solution (protein concentration: 2 mg / mL), and 1 mL of crude M8 mutant enzyme solution (protein concentration: 2 mg / mL) were added. The reaction was carried out at 70°C for 24 h. The yields of 2'-fluoro-2'-deoxyadenosine obtained under different phosphate buffer concentrations are shown in Table 8.

[0102] Table 8 Optimization of phosphate buffer concentration

[0103] Concentration / mM Yield / % 8 20.78 10 34.98 20 30.74 30 27.92 40 26.97 50 24.18

[0104] Example 12 Optimization of substrate molar ratio

[0105] Phosphate buffered saline (PBS) with a pH of 6.0 and a concentration of 10 mM was prepared, and TtPyNP and the optimal mutant M8 obtained in Example 8 were respectively processed according to the steps in Example 4 to prepare crude enzyme solutions of TtPyNP and M8 mutant, respectively.

[0106] In a 2 mL reaction system, 0.0025 g of 2'-fluoro-2'-deoxyuridine was added, and 0.000675 g, 0.0014 g, 0.0027 g, 0.0041 g, 0.0054 g, and 0.0068 g of adenine were added, respectively, with substrate molar ratios of 1:0.5, 1:1, 1:2, 1:3, 1:4, and 1:5, respectively. 1 mL of TtPyNP crude enzyme solution (protein concentration of 2 mg / mL) and 1 mL of M8 mutant crude enzyme solution (protein concentration of 2 mg / mL) were then added, and the reaction was carried out at 70°C for 24 h. The yield of 2'-fluoro-2'-deoxyadenosine was determined by high performance liquid chromatography according to the method described in Example 6. The yields of 2'-fluoro-2'-deoxyadenosine obtained under different substrate ratios are shown in Table 9.

[0107] Table 9 Substrate molar ratio optimization

[0108] Substrate molar ratio Yield / % 1:0.5 33.34 1:1 34.98 1:2 37.39 1:3 36.47 1:4 36.07 1:5 36.99

[0109] Example 13 Optimization of the Dual Enzyme Ratio

[0110] Phosphate buffered saline (PBS) with a pH of 6.0 and a concentration of 10 mM was prepared, and TtPyNP and the optimal mutant M8 obtained in Example 8 were respectively processed according to the steps in Example 4 to prepare crude enzyme solutions of TtPyNP and M8 mutant, respectively.

[0111] In a 2 mL reaction system, the substrate was 0.0025 g 2'-fluoro-2'-deoxyuridine and 0.0027 g adenine, 200 μL TtPyNP crude enzyme solution (protein concentration was 10 mg / mL) was added, and 100 μL, 200 μL, 400 μL, 600 μL, 800 μL and 1000 μL of the M8 mutant crude enzyme solution (protein concentration was 10 mg / mL) were added, respectively, that is, the dual enzyme ratios were: 1:0.5, 1:1, 1:2, 1:3, 1:4 and 1:5, respectively. The reaction was carried out at 70 ° C for 24 h, and the yield of 2'-fluoro-2'-deoxyadenosine was measured by high performance liquid chromatography according to the method described in Example 6. The yields of 2'-fluoro-2'-deoxyadenosine obtained using different dual enzyme ratios are shown in Table 10.

[0112] Table 10 Optimization of the ratio of two enzymes

[0113] Dual enzyme ratio Yield / % 1:0.5 18.3 1:1 37.39 1:2 51.96 1:3 59.42 1:4 61.3 1:5 62.69

[0114] As can be seen from Table 10, the growth rates of 1:4 and 1:5 are not large, so in subsequent experiments, 1:3 was selected because of the greater yield improvement.

[0115] Example 14 Optimization of enzyme addition amount

[0116] Phosphate buffered saline (PBS) with a pH of 6.0 and a concentration of 10 mM was prepared, and TtPyNP and the optimal mutant M8 obtained in Example 8 were respectively processed according to the steps in Example 4 to prepare crude enzyme solutions of TtPyNP and M8 mutant, respectively.

[0117] In a 2 mL reaction system, the substrates were 0.0025 g 2'-fluoro-2'-deoxyuridine and 0.0027 g adenine. As described in Example 13, the ratio of TtPyNP crude enzyme solution to M8 mutant crude enzyme solution protein content was 1:3. The amounts of TtPyNP crude enzyme solution (protein concentration of 15 mg / mL) added were 133 μL, 200 μL, 267 μL, 333 μL, and 400 μL, respectively. The added TtPyNP protein contents were 2 mg, 3 mg, 4 mg, 5 mg, and 6 mg, respectively, and the added M8 mutant protein contents were 6 mg, 9 mg, 12 mg, 15 mg, and 18 mg, respectively. The reaction was then carried out at 70°C for 24 h, and the yield of 2'-fluoro-2'-deoxyadenosine was measured using high-performance liquid chromatography as described in Example 6. The yields of 2'-fluoro-2'-deoxyadenosine obtained under different substrate ratios are shown in Table 11.

[0118] Table 11 Enzyme dosage optimization

[0119] Enzyme amount / mg Yield / % 2 59.42 3 71.4 4 73.28 5 79.02 6 80.34

[0120] As shown in Table 11, when the enzyme dosage is 6 mg, the yield is less improved than that of 5 mg, so 5 mg, which has a greater improvement, is selected as the optimal enzyme dosage.

[0121] Example 15: Determination of thermal stability of ApMTAP unmutated enzyme and M8 mutant enzyme

[0122] The ApMTAP plasmid constructed in Example 2 was prepared according to the methods of Examples 3 and 4 to obtain a crude enzyme solution, which was named ApMTAP non-mutated enzyme.

[0123] The unmutated ApMTAP enzyme and the optimal mutant M8 obtained in Example 8 were processed according to the steps in Example 4 to prepare crude enzyme solutions of ApMTAP and the M8 mutant.

[0124] The two crude enzyme solutions were incubated at 70°C, and the crude enzyme solutions of the M8 mutant and the ApMTAP unmutated enzyme were taken out at 0, 3, 6, 12, 18, and 24 hours for enzyme activity determination according to the method described in Example 5. The enzyme activity at 0 hour was set as 100%, and the test results were as follows: Figure 3 shown.

[0125] Figure 3 The enzymatic activity of ApMTAP unmutated enzyme and M8 mutant at 70℃ was measured.

[0126] Depend on Figure 3 It can be seen that after the unmutated enzyme was incubated at 70°C for 24 hours, its enzyme activity was only 57%, while the M8 mutant obtained in the present application still retained 80% of its enzyme activity after incubation at 70°C for 24 hours, indicating that the thermal stability of the 5'-methylthioadenosine phosphorylase mutant M8 of the present application is better than that of the unmutated enzyme.

[0127] Example 16 Preparation of 2'-fluoro-2'-deoxyadenosine

[0128] According to Examples 9-14, crude enzyme solutions of the M8 mutant and TtPyNP were prepared using a phosphate buffer solution with a pH of 6.0 and a concentration of 10 mM. To a 2 mL reaction system, 1 mL of the TtPyNP crude enzyme solution (protein concentration of 5 mg / mL) and 1 mL of the M8 mutant crude enzyme solution (protein concentration of 15 mg / mL) were added. The substrates were 0.0025 g of 2'-fluoro-2'-deoxyuridine and 0.0027 g of adenine. After reaction at 70° C. for 24 h, the yield of 2'-fluoro-2'-deoxyadenosine was 79.02%, as measured according to the method described in Example 6.

[0129] According to the above conditions, the yields of 2'-fluoro-2'-deoxyadenosine generated by reacting 2'-fluoro-2'-deoxyuridine at different concentrations for 24 h, 48 h and 72 h were determined. The results are shown in Table 12.

[0130] Table 12 2'-Fluoro-2'-deoxyadenosine yield

[0131]

[0132] Table 12 shows that the yield of 2'-fluoro-2'-deoxyadenosine is highest when the concentration of 2'-fluoro-2'-deoxyuridine is 5 mM. Furthermore, site-directed mutagenesis using molecular docking resulted in two rounds of screening for the optimal mutant, M8, which exhibited approximately a 12-fold increase in specific activity. 50 mM 2'-fluoro-2'-deoxyuridine produced nearly 31 mM 2'-fluoro-2'-deoxyadenosine within 72 hours, thus reducing costs and catalyst usage, making it suitable for enzyme catalysis.

[0133] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A 5'-methylthioadenosine phosphorylase mutant, characterized by: Its amino acid sequence is obtained by mutating the sequence shown in SEQ ID No. 3 in the sequence listing, and the mutation is selected from any one of the following: H64A, P131D, W114N, L206S, E168Q, F217V, F217S, F217W, R219A, R219Q, R219F, V184A and V184T. 2.5'-Methylthioadenosine phosphorylase mutant, characterized by: The amino acid sequence is obtained by mutating the sequence shown in SEQ ID No. 3 in the sequence listing, wherein the mutation is selected from at least two of the following: H64A, P131D, W114N and L206S.

3. The 5'-methylthioadenosine phosphorylase mutant according to claim 2, characterized in that: The mutation is: H64A+W114N+L206S. A polynucleotide encoding the 5'-methylthioadenosine phosphorylase mutant according to any one of claims 1 to 3.

5. The polynucleotide encoding a 5'-methylthioadenosine phosphorylase mutant according to claim 4, characterized in that: The polynucleotide sequence is shown as SEQ ID NO.

6.

6. A recombinant expression vector comprising the polynucleotide according to claim 4 or 5.

7. The recombinant expression vector according to claim 6, characterized in that: The recombinant expression vector comprises pET-28a(+).

8. A genetically engineered bacterium comprising the recombinant expression vector according to claim 6 or 7.

9. The genetically engineered bacterium according to claim 8, characterized in that: The genetically engineered bacteria is a recombinant strain obtained by connecting the polynucleotide of claim 4 or 5 to a vector to obtain a recombinant expression vector, and then introducing it into a host bacterium for inducing expression, wherein the host bacterium includes Escherichia coli, Bacillus subtilis or yeast.

10. The genetically engineered bacterium according to claim 9, characterized in that: The Escherichia coli is the Escherichia coli BL21 (DE3) strain.

11. Use of the 5'-methylthioadenosine phosphorylase mutant according to any one of claims 1 to 3, the polynucleotide according to claim 4 or 5, the recombinant expression vector according to claim 6 or 7, or the genetically engineered bacterium according to any one of claims 8 to 10 in catalyzing the synthesis of 2'-fluoro-2'-deoxyadenosine.

12. The use according to claim 11, characterized in that: 2'-fluoro-2'-deoxyuridine, adenine, pyrimidine nucleoside phosphorylase TtPyNP and the 5'-methylthioadenosine phosphorylase mutant according to any one of claims 1 to 3 are added to the reaction system, and the reaction is carried out at 50-90°C.

13. The use according to claim 12, characterized in that: The crude enzyme solution of the mutant and TtPyNP as claimed in claim 3 was prepared using phosphate buffer with a pH of 5.5-8.5 and a concentration of 8-50 mM.

14. The use according to claim 13, characterized in that: The crude enzyme solution of the mutant and TtPyNP as claimed in claim 3 was prepared using phosphate buffer with a pH of 6.0 and a concentration of 10 mM.

15. The use according to claim 12, characterized in that: The molar ratio of the 2'-fluoro-2'-deoxyuridine to adenine is 1:0.5-1:

5.

16. The use according to claim 15, characterized in that: The molar ratio of the 2'-fluoro-2'-deoxyuridine to adenine is 1:

2.

17. The use according to claim 12, characterized in that: The amount of 2'-fluoro-2'-deoxyuridine added is 5-50 mM.

18. The use according to claim 17, characterized in that: The amount of 2'-fluoro-2'-deoxyuridine added is 30-50 mM.

19. The use according to claim 12, characterized in that: The protein weight ratio of the pyrimidine nucleoside phosphorylase TtPyNP and the 5'-methylthioadenosine phosphorylase mutant is 1:0.5-1:

5.

20. The use according to claim 19, characterized in that: The protein weight ratio of the pyrimidine nucleoside phosphorylase TtPyNP and the 5'-methylthioadenosine phosphorylase mutant is 1:

3.

21. The use according to claim 12, characterized in that: In the reaction system, the protein content of the pyrimidine nucleoside phosphorylase TtPyNP is 1-3 mg / mL, and the protein content of the 5'-methylthioadenosine phosphorylase mutant is 3-9 mg / mL.

22. The use according to claim 21, characterized in that: In the reaction system, the protein content of the pyrimidine nucleoside phosphorylase TtPyNP was 2.5 mg / mL, and the protein content of the 5'-methylthioadenosine phosphorylase mutant was 7.5 mg / mL.

23. The use according to claim 12, characterized in that: The reaction time is 1-96h.

24. The use according to claim 23, characterized in that: The reaction time is 24-72h.

25. The use according to claim 12, characterized in that: The reaction temperature was 70°C.

26. A method for producing 2'-fluoro-2'-deoxyadenosine, characterized in that: 2'-fluoro-2'-deoxyuridine, adenine, pyrimidine nucleoside phosphorylase TtPyNP and the 5'-methylthioadenosine phosphorylase mutant according to any one of claims 1 to 3 are added to the reaction system, and the reaction is carried out at 50-90°C.

27. The method for producing 2'-fluoro-2'-deoxyadenosine according to claim 26, characterized in that: The crude enzyme solution of the mutant and TtPyNP as claimed in claim 3 was prepared using phosphate buffer with a pH of 5.5-8.5 and a concentration of 8-50 mM.

28. The method for producing 2'-fluoro-2'-deoxyadenosine according to claim 27, characterized in that: The crude enzyme solution of the mutant and TtPyNP as claimed in claim 3 was prepared using phosphate buffer with a pH of 6.0 and a concentration of 10 mM.

29. The method for producing 2'-fluoro-2'-deoxyadenosine according to claim 26, wherein: The molar ratio of the 2'-fluoro-2'-deoxyuridine to adenine is 1:0.5-1:

5.

30. The method for producing 2'-fluoro-2'-deoxyadenosine according to claim 29, characterized in that: The molar ratio of the 2'-fluoro-2'-deoxyuridine to adenine is 1:

2.

31. The method for producing 2'-fluoro-2'-deoxyadenosine according to claim 26, wherein: The amount of 2'-fluoro-2'-deoxyuridine added is 5-50 mM.

32. The method for producing 2'-fluoro-2'-deoxyadenosine according to claim 31, characterized in that: The amount of 2'-fluoro-2'-deoxyuridine added is 30-50 mM.

33. The method for producing 2'-fluoro-2'-deoxyadenosine according to claim 26, wherein: The protein weight ratio of the pyrimidine nucleoside phosphorylase TtPyNP and the 5'-methylthioadenosine phosphorylase mutant is 1:0.5-1:

5.

34. The method for producing 2'-fluoro-2'-deoxyadenosine according to claim 33, wherein: The protein weight ratio of the pyrimidine nucleoside phosphorylase TtPyNP and the 5'-methylthioadenosine phosphorylase mutant is 1:

3.

35. The method for producing 2'-fluoro-2'-deoxyadenosine according to claim 26, wherein: In the reaction system, the protein content of the pyrimidine nucleoside phosphorylase TtPyNP is 1-3 mg / mL, and the protein content of the 5'-methylthioadenosine phosphorylase mutant is 3-9 mg / mL.

36. The method for producing 2'-fluoro-2'-deoxyadenosine according to claim 35, characterized in that: In the reaction system, the protein content of the pyrimidine nucleoside phosphorylase TtPyNP was 2.5 mg / mL, and the protein content of the 5'-methylthioadenosine phosphorylase mutant was 7.5 mg / mL.

37. The method for producing 2'-fluoro-2'-deoxyadenosine according to claim 26, wherein: The reaction time is 1-96h.

38. The method for producing 2'-fluoro-2'-deoxyadenosine according to claim 37, wherein: The reaction time is 24-72h.

39. The method for producing 2'-fluoro-2'-deoxyadenosine according to claim 26, wherein: The reaction temperature was 70°C.

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