5 '-methylthio adenosine phosphorylase mutant and application thereof

Abstract

The invention provides a 5 '-methylthio adenosine phosphorylase mutant, the amino acid sequence of the 5'-methylthio adenosine phosphorylase mutant is obtained by mutating a sequence as shown in SEQ ID No.3 in a sequence table, and the mutation is selected from at least one of the following components: H64A, P131D, W114N, L206S, E168Q, F217V, F217S, F217W, R219A, R219Q, R219F, V184A and V184T. The specific enzyme activity of the mutant H64A / W114N / L206S reaches 1.31 U / g, the specific enzyme activity of an ApMTAP non-mutated enzyme strain is 0.11 U / g, and the specific enzyme activity is improved by about 12 times, that is, the yield of 2 '-fluoro-2'-deoxyadenosine is greatly improved, and the mutant H64A / W114N / L206S is suitable for the field of enzyme catalysis.

Description

Technical Field

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

[0002] Nucleosides and their analogs have been used as small molecule drugs and are widely used in cancer and antiviral treatment, including but not limited to valacyclovir for the treatment of HSV-1, cytarabine for the treatment of special leukemias, etc. People's interest in nucleoside analogs has greatly increased, and a variety of nucleoside analogs or combination drugs have been approved for the treatment of a variety of 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 interest. Among them, substitution of hydrogen or hydroxyl groups in the pentose moiety of nucleosides with fluorine has produced analogs with a variety of biological activities. 2'-Fluorinated nucleosides have been shown to have good properties as components of antisense oligonucleotides and small interfering RNAs, and have better efficacy in disease treatment than other C2'-modified nucleotides. Nucleoside analogs with 2'-deoxy-2'-fluororibosyl moieties have been studied and show activity against herpes simplex virus, pseudorabies virus, equine abortion virus, influenza virus, and varicella virus, and some also inhibit the growth of leukemia cells.

[0004] The importance of nucleoside analogs has prompted researchers to further investigate 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. The lack of enzymes with broad substrate specificity limits the scope of application of this strategy. In addition, when operated at high temperatures, the efficiency of this process is usually reduced due to the thermal instability of biocatalysts. 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, so 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] In order to solve the problems existing in the prior art, the present invention provides a 5'-methylthioadenosine phosphorylase mutant and its application, and specifically relates to its application in catalytic synthesis of 2'-fluoro-2'-deoxyadenosine. The present invention molecularly transforms the wild-type 5'-methylthioadenosine phosphorylase ApMTAP through site-directed mutagenesis technology, solves the problems of lack of substrate specificity and instability, and synthesizes 2'-fluoro-2'-deoxyadenosine by dual enzyme cascade catalysis with pyrimidine nucleoside phosphorylase TtPyNP, thereby improving the enzyme activity and the yield of 2'-fluoro-2'-deoxyadenosine.

[0006] The first object of the present invention is to provide a 5'-methylthioadenosine phosphorylase mutant, whose amino acid sequence is obtained by mutating the sequence shown in SEQ ID No. 3 in the sequence table, and 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, and then introducing it into a host bacterium for inducing 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 phosphate buffer with a pH of 5.5-8.5 and a concentration of 8-50 mM is used to prepare a crude enzyme solution of the M8 mutant and TtPyNP; more preferably, a phosphate buffer with a pH of 6.0 and a concentration of 10 mM is used to prepare a crude enzyme solution of the M8 mutant and TtPyNP;

[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, and more preferably the reaction time is 24-72h;

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

[0025] The sixth object of the present invention is to provide a method for producing 2'-fluoro-2'-deoxyadenosine, wherein 2'-fluoro-2'-deoxyuridine, adenine, pyrimidine nucleoside phosphorylase TtPyNP and the above-mentioned 5'-methylthioadenosine phosphorylase mutant are added to the reaction system, and the reaction is carried out at 50-90°C;

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

[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, and 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 2'-fluoro-2'-deoxyuridine to make it split into phosphorylated 2'-fluoropentose and free uracil, and then utilizes 5'-methylthioadenosine phosphorylase to catalyze the intermediate product phosphorylated 2'-fluoropentose to combine with adenine to prepare 2'-fluoro-2'-deoxyadenosine.

[0034] The present invention improves the catalytic performance of 5'-methylthioadenosine phosphorylase (ApMTAP) from Aeropyrumpernix K1 by semi-rational design, and screens out the optimal triple mutant H64A / W114N / L206S with a specific enzyme activity of 1.31 U / g, while the ApMTAP unmutated enzyme strain has a specific enzyme activity of 0.11 U / g, which is about 12 times higher, that is, the yield of 2'-fluoro-2'-deoxyadenosine is greatly improved, and the present invention 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 This is a 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 without mutant enzyme and M8 mutant at 70℃. DETAILED DESCRIPTION

[0039] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are 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, which 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. It 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 well. 5 μL was added to 50 μL of E. coli Bl21 (DE3) competent cells, flicked to mix well, placed on ice for 30 minutes, placed in a 42°C water bath for heat shock for 45 seconds, and added to 500 μL of sterile, non-resistant LB liquid culture medium after ice bathing for 2 minutes. After mixing, place on a 37°C shaker for activation for 1 hour. Then centrifuge at 10000 rpm for 1 minute, take out 300 μL of supernatant, mix the remaining liquid with the bacterial sediment, take 50 μL and apply it to a LB solid culture medium plate containing kanamycin, and culture 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 added to 10 mL of LB liquid medium containing kanamycin. After culturing 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 cultured at 37°C for 16 h. IPTG was added at a final concentration of 0.5 mM to induce expression, and the induction was performed at 20°C for 18 h. The bacterial solution at the end of the culture was centrifuged at 8000 rpm for 5 min to collect the bacterial pellet, and stored at -20°C for later use.

[0056] Preparation of enzyme solution: resuspend the above bacterial pellet with 50 mL of PBS, and then use a cell disruptor to disrupt the cells evenly at a pressure of 700 to 900 bar for 45 seconds. After the bacterial solution is completely disrupted, centrifuge it at 12,000 rpm for 15 minutes, collect the supernatant, and remove the remaining cell debris.

[0057] Example 5 Enzyme activity determination

[0058] In a 2mL reaction system, add 0.00049g of 2'-fluoro-2'-deoxyuridine and 0.00054g of adenine. Add only 1mL of TtPyNP crude enzyme solution to react for 4h under PBS 10mM, pH 6.0, 70℃ to make the intermediate product phosphorylated 2'-fluoropentose sufficient, and then add 1mL of 5'-methylthioadenosine phosphorylase crude enzyme solution to react for 1h. The reaction solution was filtered and the yield of 2'-fluoro-2'-deoxyadenosine was measured by high performance liquid chromatography. Enzyme activity is defined as: 1U is 1μM of 2'-fluoro-2'-deoxyadenosine generated within 1min.

[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 elution time of uracil was about 3.5 min, the elution time of adenine was about 4.3 min, the elution time of 2'-fluoro-2'-deoxyuridine was about 4.6 min, and the elution time of 2'-fluoro-2'-deoxyadenosine was about 8.0 min. The experimental results are shown in Figure 1 .

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

[0062] Example 7 Preliminary 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 non-mutated 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, and performs molecular docking with the substrate adenine and the intermediate product 1'-phospho-2'-fluoropentose on pymol. After the docking, the surrounding amino acids and other amino acids that may affect the catalytic performance are site-directed mutagenesis. The initial screening results of 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] The ApMTAP non-mutated enzyme plasmid was used as a template, and PCR amplification was performed using the designed primers and KOD DNA polymerase. After verification by agarose gel electrophoresis, DpnⅠ restriction enzyme was added to the PCR reaction product to remove the template. The final reaction product was then transformed into Escherichia coli BL21 (DE3) according to the method in Example 3, and the mutant crude enzyme solution was obtained by the method in Example 4. The relative activity was measured using liquid phase, and finally four optimal single-point mutants were screened out: H64A, W114N, P131D and L206S.

[0066] It can also be handed over to a biological company to directly synthesize each gene sequence containing 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 to perform site-directed mutagenesis. The site-directed mutagenesis PCR reaction system is shown in Table 3, and the PCR reaction program is shown in Table 4.

[0075] Table 3 PCR reaction system

[0076] PCR reaction system Volume / μL Sterile water 38 KODOneTMPCRMasterMix 50 Plasmids 6 Upstream primer 3 Downstream primer 3

[0077] Table 4 PCR reaction program

[0078]

[0079] Example 85'-Methylthioadenosine phosphorylase mutant rescreening

[0080] After screening out the four most active single mutants, they were 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 shown 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] It can be seen from Table 5 that the best mutant is M8.

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

[0085] MRKPVHLEAGPGDVAPLVVAVGDPGRAERLATGLLEDARLVSSARGLKVYTGSFNGSEVTIATAGIGGPSAAVVFEELRMLGAEVLVRLGTSGGLSKDLRLGDVVVAAGAGCYNGSGGSIQY AGERPMCLPASPDPILTAGIYRGLSSRLGDRVVLAPVMSSDAFYAETPEAAGRWRSLLGMAAVEMELHTLFSISWIRGFRSAGVSIVSDLLLPEGFKRITPGELARREVEVGRALLEVLTGGV

[0086] The 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 operated 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 2mL reaction system, 0.0025g 2'-fluoro-2'-deoxyuridine and 0.0027g adenine, 1mL TtPyNP crude enzyme solution (protein concentration of 2mg / mL) and 1mL M8 mutant crude enzyme solution (protein concentration of 2mg / 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 24h. According to the method described in Example 6, the yield of 2'-fluoro-2'-deoxyadenosine was measured using high performance liquid chromatography. Under different reaction temperature conditions, the yield of 2'-fluoro-2'-deoxyadenosine obtained is 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] In a 2mL reaction system, 0.0025g 2'-fluoro-2'-deoxyuridine and 0.0027g adenine, 1mL TtPyNP crude enzyme solution (protein concentration of 2mg / mL) and 1mL M8 mutant crude enzyme solution (protein concentration of 2mg / mL) were added, and the reaction was carried out at 70°C for 24h. According to the method described in Example 6, the yield of 2'-fluoro-2'-deoxyadenosine was measured by high performance liquid chromatography. Under phosphate buffer conditions of different pH, the yields of 2'-fluoro-2'-deoxyadenosine obtained 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 different concentrations of 8 mM, 10 mM, 20 mM, 30 mM, 40 mM and 50 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 mutants with different concentrations.

[0101] 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 70°C for 24 h. The yields of 2'-fluoro-2'-deoxyadenosine obtained under different concentrations of phosphate buffer 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 Substrate molar ratio optimization

[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 operated according to the steps in Example 4 to prepare crude enzyme solutions of TtPyNP and M8 mutant, respectively.

[0106] In a 2mL reaction system, 0.0025g 2'-fluoro-2'-deoxyuridine was added, and 0.000675g, 0.0014g, 0.0027g, 0.0041g, 0.0054g and 0.0068g of adenine were added, and the substrate molar ratios were 1:0.5, 1:1, 1:2, 1:3, 1:4 and 1:5, respectively. Then 1mL of TtPyNP crude enzyme solution (protein concentration of 2mg / mL) and 1mL of M8 mutant crude enzyme solution (protein concentration of 2mg / mL) were added, and the reaction was carried out at 70°C for 24h. According to the method described in Example 6, the yield of 2'-fluoro-2'-deoxyadenosine was measured by high performance liquid chromatography. Under different substrate ratio conditions, the yield of 2'-fluoro-2'-deoxyadenosine obtained is 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 ratio of two enzymes

[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 operated according to the steps in Example 4 to prepare crude enzyme solutions of TtPyNP and M8 mutant, respectively.

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

[0112] Table 10 Optimization of the ratio of double 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] It can be seen from Table 10 that the growth rates of 1:4 and 1:5 are not large, so 1:3 with a greater increase in yield was selected in subsequent experiments.

[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 operated according to the steps in Example 4 to prepare crude enzyme solutions of TtPyNP and M8 mutant, respectively.

[0117] In a 2mL reaction system, the substrate is 0.0025g 2'-fluoro-2'-deoxyuridine and 0.0027g adenine. According to Example 13, the ratio of the protein content of the crude enzyme solution of TtPyNP to the crude enzyme solution of M8 mutant is 1:3, and the amount of crude enzyme solution of TtPyNP (protein concentration is 15mg / mL) added is 133μL, 200μL, 267μL, 333μL and 400μL, that is, the protein content of TtPyNP added is 2mg, 3mg, 4mg, 5mg and 6mg, respectively, and the protein content of the corresponding mutant added is 6mg, 9mg, 12mg, 15mg and 18mg, respectively. After that, the reaction is carried out at 70°C for 24h, and the yield of 2'-fluoro-2'-deoxyadenosine is measured by high performance liquid chromatography according to the method described in Example 6. Under different substrate ratio conditions, the yields of 2'-fluoro-2'-deoxyadenosine obtained 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] It can be seen from Table 11 that when the enzyme dosage is 6 mg, the yield improvement is less than that of 5 mg, so 5 mg, which has a greater improvement, is selected as the optimal enzyme dosage.

[0121] Example 15: Thermal stability test of ApMTAP unmutated enzyme and M8 mutant enzyme

[0122] The ApMTAP plasmid constructed in Example 2 was used according to the methods of Examples 3 and 4 to prepare 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 mutant M8.

[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 to perform enzyme activity assays 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 without mutant enzyme and M8 mutant at 70℃.

[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 having a pH of 6.0 and a concentration of 10 mM. In 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 reacting at 70° C. for 24 h, the yield of the obtained 2'-fluoro-2'-deoxyadenosine was 79.02% as measured by the method shown 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 24h, 48h and 72h were determined. The results are shown in Table 12.

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

[0131]

[0132] As shown in Table 12, when the concentration of 2'-fluoro-2'-deoxyuridine is 5 mM, the yield of 2'-fluoro-2'-deoxyadenosine is the highest. It can also be seen that by molecular docking for site-directed mutagenesis, the optimal mutant M8 has a specific activity increased by about 12 times after two rounds of screening, and 50 mM 2'-fluoro-2'-deoxyuridine reacts for 72 hours to generate nearly 31 mM 2'-fluoro-2'-deoxyadenosine, thereby reducing the required cost and the amount of catalyst used, which is suitable for the field of enzyme catalysis.

[0133] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is 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 can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. 5'-methylthioadenosine phosphorylase mutant, characterized in that: Its amino acid sequence is obtained by mutating the sequence shown in SEQ ID No. 3 in the sequence table, and 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.

2. The 5'-methylthioadenosine phosphorylase mutant according to claim 1, characterized in that: The mutations are selected from at least two of the following: H64A, P131D, W114N and L206S; Preferably, the mutation is: H64A+W114N+L206S.

3. A nucleotide sequence encoding the 5'-methylthioadenosine phosphorylase mutant according to claim 1 or 2.

4. The nucleotide sequence encoding the 5'-methylthioadenosine phosphorylase mutant according to claim 3, characterized in that: The nucleotide sequence is SEQ ID NO.

6.

5. A recombinant expression vector comprising the nucleotide sequence of claim 3 or 4; Preferably, the recombinant expression vector comprises pET-28a(+).

6. A genetically engineered bacterium comprising the recombinant expression vector according to claim 4; Preferably, the genetically engineered bacteria is a recombinant strain obtained by connecting the nucleotide sequence of claim 3 or 4 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; Preferably, the Escherichia coli is the Escherichia coli BL21 (DE3) strain.

7. Use of the 5'-methylthioadenosine phosphorylase mutant according to claim 1 or 2, the nucleotide sequence according to claim 3 or 4, the recombinant expression vector according to claim 5, and the genetically engineered bacteria according to claim 6 in catalyzing the synthesis of 2'-fluoro-2'-deoxyadenosine.

8. The use according to claim 7, characterized in that: Add 2'-fluoro-2'-deoxyuridine, adenine, pyrimidine nucleoside phosphorylase TtPyNP and the 5'-methylthioadenosine phosphorylase mutant according to claim 1 or 2 into the reaction system, and react at 50-90° C.; Preferably, a phosphate buffer with a pH of 5.5-8.5 and a concentration of 8-50 mM is used to prepare a crude enzyme solution of the M8 mutant and TtPyNP; more preferably, a phosphate buffer with a pH of 6.0 and a concentration of 10 mM is used to prepare a crude enzyme solution of the M8 mutant and TtPyNP; 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; 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; 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; 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; Preferably, the reaction time is 1-96h, and more preferably the reaction time is 24-72h; Preferably, the reaction temperature is 70°C.

9. A method for producing 2'-fluoro-2'-deoxyadenosine, characterized in that: Add 2'-fluoro-2'-deoxyuridine, adenine, pyrimidine nucleoside phosphorylase TtPyNP and the 5'-methylthioadenosine phosphorylase mutant according to claim 1 or 2 into the reaction system, and react at 50-90° C.; Preferably, a phosphate buffer with a pH of 5.5-8.5 and a concentration of 8-50 mM is used to prepare a crude enzyme solution of the M8 mutant and TtPyNP; more preferably, a phosphate buffer with a pH of 6.0 and a concentration of 10 mM is used to prepare a crude enzyme solution of the M8 mutant and TtPyNP; 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; 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; 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; 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; Preferably, the reaction time is 1-96h, and more preferably the reaction time is 24-72h; Preferably, the reaction temperature is 70°C.

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