Method for synthesizing tetrahydrofolic acid based on enzyme microenvironment regulation
By expressing fusion dihydrofolate reductase and superfolded green fluorescent protein in Escherichia coli BL21 (DE3), an enzyme microenvironment regulation system was constructed, and the problem of mismatch in enzyme catalytic conditions in tetrahydrofolate synthesis was solved, and efficient tetrahydrofolate production was achieved.
Patent Information
- Application Number
- CN202510582261.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-07
- Publication Date
- 2025-08-08
AI Technical Summary
The prior art has problems of high cost, complex operation and poor product purity and yield in tetrahydrofolate synthesis, especially chemical reduction methods and catalytic hydrogenation methods. The biological enzymatic method still needs to be improved in the optimization of enzyme catalytic conditions.
E. coli BL21 (DE3) was used to express fusion dihydrofolate reductase and superfolded green fluorescent protein, and the local microenvironment of the enzyme was adjusted through covalent ligation to construct an enzyme cascade system with self-sufficiency of NADPH cofactors to optimize catalytic performance.
The synthesis efficiency of tetrahydrofolate was significantly improved, with the yield reaching 3270.1μM at pH 7.0 and 4223.4μM at pH 5.0, which solved the problem of mismatch in enzyme catalytic conditions and provided a new method for industrial production.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biocatalysis, and in particular to a method for synthesizing tetrahydrofolate based on enzyme microenvironment regulation. Background Art
[0002] Tetrahydrofolate, the biologically active form of folate, plays a vital role in the body. It acts as a transporter for one-carbon groups and is essential in the biosynthesis of amino acids (such as glycine, methionine, and its derivative formylmethionine) and nucleic acid components (including thymidylate, pantothenic acid, and purine nucleotides). This series of biochemical processes is crucial for maintaining normal physiological functions. However, when folate supply is insufficient or the activity of key enzymes involved in folate metabolism, such as dihydrofolate reductase and methylenetetrahydrofolate reductase, is blocked, the conversion of folate to the physiologically active L-5-methyltetrahydrofolate is disrupted, potentially leading to a range of health problems, including serious conditions such as neural tube defects, megaloblastic anemia, increased cancer risk, and DNA damage. Given its importance, tetrahydrofolate has been widely used in the medical and healthcare fields. It is used directly as an anti-anemia drug to treat anemia, and it also plays a synergistic role in cancer treatment, enhancing the inhibition of thymidylate synthase by fluorouracil, improving the efficiency of chemotherapy and aiding in the elimination of tumor cells. In addition, tetrahydrofolate and its related compounds also show great application potential in agriculture and food industries, further highlighting the necessity of their research and development.
[0003] Researchers have explored a variety of methods for the synthesis of tetrahydrofolate. The main methods reported in the literature include catalytic hydrogenation, chemical reduction, and enzymatic methods (specifically, the dihydrofolate reductase method). The catalytic hydrogenation method is limited by its high cost, complex operation process, and the use of a large number of metal catalysts. Although the chemical reduction method is the mainstream method for industrial preparation, it faces the challenges of frequent side reactions caused by the strong alkalinity of the reducing agent, poor product purity, and yield. In contrast, the dihydrofolate reductase method relies on the high selectivity of biological enzymes and provides a more ideal synthesis route, but it still needs to overcome difficulties such as the optimization of enzyme catalytic conditions. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to address the deficiencies of the existing technology and provide a method for synthesizing tetrahydrofolate based on enzyme microenvironment regulation.
[0005] In order to solve the above technical problems, the present invention discloses a method for synthesizing tetrahydrofolate based on enzyme microenvironment regulation, which comprises the following steps: using Escherichia coli BL21 (DE3) as a host cell to express a fusion dihydrofolate reductase, a fusion superfolded green fluorescent protein and a formate dehydrogenase in biocatalysis, using TB medium for fermentation and culture, ultrasonically disrupting the cells and purifying them to obtain a corresponding pure enzyme solution; dihydrofolate reductase catalyzes the conversion of folic acid into tetrahydrofolate and consumes the NADPH cofactor, while the formate dehydrogenase converts NADP into tetrahydrofolate in the reaction of oxidizing sodium formate. + Reduced to NADPH; The present invention conjugates dihydrofolate reductase with superfolded green fluorescent protein to adjust the local microenvironment of the enzyme, thereby optimizing its catalytic performance and significantly increasing the production of tetrahydrofolate. The specific technical solution is as follows:
[0006] A method for synthesizing tetrahydrofolate based on enzyme microenvironment regulation, using folic acid as a substrate and NADPH as a cofactor, and catalyzing the synthesis of tetrahydrofolate by fusion of superfolded green fluorescent protein and fusion of dihydrofolate reductase;
[0007] Wherein, the catalytic reaction system includes: folic acid, sodium formate, NADP + , fusion superfolded green fluorescent protein-fusion dihydrofolate reductase, formate dehydrogenase, buffer; the NADP + Reduced to NADPH by formate dehydrogenase.
[0008] The fusion superfolded green fluorescent protein-fusion dihydrofolate reductase (sfGFP-6GS-SmDHFR) is obtained by mixing fusion superfolded green fluorescent protein (sfGFP-SpyTag) and fusion dihydrofolate reductase (SpyCatcher-SmDHFR) at equimolar concentrations at 4°C for 8 to 12 hours.
[0009] Wherein, the amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO.3.
[0010] The fused dihydrofolate reductase is dihydrofolate reductase SmDHFR fused with SpyCatcher protein gene, and the fused superfolded green fluorescent protein is superfolded green fluorescent protein sfGFP fused with SpyTag protein gene.
[0011] The fusion dihydrofolate reductase is obtained by cloning a fragment of the dihydrofolate reductase SmDHFR gene folA and a gene fragment of the SpyCatcher protein into an expression vector, then introducing the vector into Escherichia coli to induce expression, obtain cells, and ultrasonically disrupt and purify the cells.
[0012] Specifically, the expression vector is pRSFDuet-1.
[0013] The fusion super-folded green fluorescent protein is obtained by introducing a recombinant plasmid containing the super-folded green fluorescent protein gene and the SpyTag protein gene into Escherichia coli to induce expression, and then ultrasonically disrupting and purifying the cells.
[0014] Specifically, the recombinant plasmid was synthesized by General Biotechnology Company.
[0015] Among them, the amino acid sequence of the SpyCatcher protein is shown in SEQ ID NO.5; the amino acid sequence of the dihydrofolate reductase is shown in SEQ ID NO.1; and the amino acid sequence of the SpyTag protein is shown in SEQ ID NO.13.
[0016] The superfolded green fluorescent protein includes any one of a superfolded green fluorescent protein with 30 negative charges (-30sfGFP), an uncharged superfolded green fluorescent protein (0sfGFP), and a superfolded green fluorescent protein with 36 positive charges (+36sfGFP).
[0017] Specifically, the amino acid sequence of the superfolded green fluorescent protein with 30 negative charges is shown in SEQ ID NO.7; the amino acid sequence of the uncharged superfolded green fluorescent protein is shown in SEQ ID NO.8; and the amino acid sequence of the superfolded green fluorescent protein with 36 positive charges is shown in SEQ ID NO.9.
[0018] The conditions for the catalytic synthesis are as follows: under anaerobic conditions, the reaction temperature is 35-40° C., the reaction pH is 5.0-7.0, and the reaction time is 2-17 hours.
[0019] In some embodiments of the present invention, the catalytic synthesis is carried out under the following conditions: under anaerobic conditions, the reaction temperature is 37° C., the reaction pH is 5.0 or 7.0, and the reaction time is 2 to 17 hours.
[0020] Wherein, in the catalytic reaction system, the concentration of folic acid is 0.1-50 mM, the concentration of sodium formate is 6-60 mM, and the concentration of NADP is 0.1-50 mM. + The concentration of is 1-10 mM, the concentration of fusion superfolded green fluorescent protein-fusion dihydrofolate reductase is 10-100 μM, and the concentration of formate dehydrogenase is 2-20 μM.
[0021] In some embodiments of the present invention, in the catalytic reaction system, the concentration of folic acid is 20mM, the concentration of sodium formate is 60mM, and the concentration of NADP is 20mM. +The concentration of was 10 mM, the concentration of superfolded green fluorescent protein-fused dihydrofolate reductase was 100 μM, and the concentration of formate dehydrogenase was 10 μM.
[0022] Wherein, the buffer is 50-200 mM phosphate buffer with a pH of 6.0-7.0 or 50-200 mM acetate buffer with a pH of 5.0.
[0023] Specifically, when the pH of the catalytic synthesis is 6.0-7.0, the buffer is 50-200 mM phosphate buffer at pH 6.0-7.0; when the pH of the catalytic synthesis is 5.0, the buffer is 50-200 mM acetate buffer at pH 5.0.
[0024] In some embodiments of the present invention, when the pH of the catalytic synthesis is 6.0-7.0, the buffer is 200 mM pH 6.0-7.0 phosphate buffer; when the pH of the catalytic synthesis is 5.0, the buffer is 200 mM pH 5.0 acetate buffer.
[0025] Furthermore, the method specifically comprises the following steps:
[0026] (1) The gene fragments encoding SmDHFR and SpyCatcher were cloned into the pRSFDuet plasmid to construct the recombinant expression vector pRSFDuet-SpyCatcher-folA; the recombinant plasmids pRSFDuet-sfGFP-SpyTag (pRSFDuet-(-30)sfGFP-SpyTag, pRSFDuet-(0)sfGFP-SpyTag, and pRSFDuet-(+36)sfGFP-SpyTag) were constructed by Shanghai General Biotechnology.
[0027] (2) The recombinant expression vector pRSFDuet-SpyCatcher-folA is introduced into Escherichia coli, cultured in TB liquid medium and added with 0.75mM IPTG to induce protein expression to obtain cells, which are ultrasonically disrupted and purified to obtain a pure enzyme solution fused with dihydrofolate reductase; the recombinant expression vector pRSFDuet-sfGFP-SpyTag is introduced into Escherichia coli, cultured in TB liquid medium and added with 0.75mM IPTG to induce protein expression to obtain cells, which are ultrasonically disrupted and purified to obtain a pure enzyme solution fused with superfolded green fluorescent protein; preferably, the Escherichia coli is Escherichia coli BL21 (DE3).
[0028] (3) The pure enzyme solution of the fusion dihydrofolate reductase prepared in step (2) and the pure enzyme solution of the fusion superfolded green fluorescent protein were mixed at equimolar concentrations at 4° C. for 8 to 12 hours to obtain a fusion superfolded green fluorescent protein-fusion dihydrofolate reductase.
[0029] (4) Folic acid, sodium formate, NADP + , formate dehydrogenase, and fused superfolded green fluorescent protein-fused dihydrofolate reductase are mixed in phosphate buffer and acetate buffer, respectively, to carry out a catalytic reaction; the catalytic reaction is carried out under anaerobic conditions, at a reaction temperature of 35-40°C, and for a reaction time of 2-17 hours. Preferably, the catalytic reaction system is: 0.1-50 mM folic acid, 6-60 mM sodium formate, 1-10 mM NADP + , 10-100 μM fused superfolded green fluorescent protein-fused dihydrofolate reductase, 2-20 μM formate dehydrogenase enzyme, 50-200 mM pH 5.0 acetate buffer or pH 6.0-7.0 phosphate buffer.
[0030] Beneficial effects:
[0031] (1) In order to optimize the biosynthesis process of tetrahydrofolate, the present invention innovatively adopts in vitro enzyme catalysis technology to construct an NADPH cofactor self-sufficient enzyme cascade system integrating dihydrofolate reductase and formate dehydrogenase, that is, dihydrofolate reductase is used to convert folic acid into tetrahydrofolate, and formate dehydrogenase is used to convert the NADP produced by the reaction into NADPH. + This system not only improves the efficiency of tetrahydrofolate synthesis, but also solves the problem of mismatched optimal pH values between different enzymes.
[0032] (2) The present invention successfully adjusts the local microenvironment of the enzyme by conjugating dihydrofolate reductase with superfolded green fluorescent protein, optimizes its catalytic performance, enhances the dual enzyme synergy of dihydrofolate reductase and formate dehydrogenase, solves the problem of inconsistent optimal pH for dihydrofolate reductase and formate dehydrogenase, improves the biosynthesis of tetrahydrofolate, and achieves a significant increase in tetrahydrofolate production. Under the catalytic condition of pH 7.0, its production reached 3270.1μM; under the catalytic condition of pH 5.0, its production reached 4223.4μM. This innovative method not only provides a new perspective for the industrial production of tetrahydrofolate, but also lays a solid foundation for the development of related biosynthesis technologies. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] The present invention will be further described in detail below with reference to the accompanying drawings, and the above and / or other advantages of the present invention will become more apparent.
[0034] Figure 1This is the verification diagram of SDS-polyacrylamide gel electrophoresis, protein marker (M).
[0035] Figure 2 Yield plots to investigate the effect of covalent conjugation of dihydrofolate reductase to superfolded green fluorescent protein on enzyme catalysis. (a) Comparison of tetrahydrofolate yields after 2 hours of reaction for SmDHFR, SpyCatcher-SmDHFR, +36sfGFP-6GS-SmDHFR, 0sfGFP-6GS-SmDHFR, and -30sfGFP-6GS-SmDHFR in pH 5.0, pH 6.0, and pH 7.0 buffer solutions. (b) Comparison of tetrahydrofolate yields after 17 hours of reaction for SmDHFR, SpyCatcher-SmDHFR, and -30sfGFP-S6GS-SmDHFR in pH 5.0 buffer solution and -30sfGFP-6GS-SmDHFR in pH 7.0 buffer solution. SmDHFR is dihydrofolate reductase, SpyCatcher-SmDHFR is dihydrofolate reductase with SpyCatcher connected to its N-terminus, and (+36, 0, -30)sfGFP-6GS-SmDHFR is dihydrofolate reductase covalently linked to superfolded green fluorescent protein via SpyCatcher / SpyTag.
[0036] Figure 3 Schematic diagram of dihydrofolate reductase and superfolded green fluorescent protein. (a) shows the electrostatic surface potential of SmDHFR (AF-A0A3E2EFG9-F1-model_v4) and (-30)sfGFP (PDB ID 2B3P). (b) I shows a schematic diagram of SmDHFR in pH 5.0 buffer; II shows a schematic diagram of SmDHFR in pH 7.0 buffer; III shows a schematic diagram of the ionic strength of -30sfGFP-6GS-SmDHFR in pH 7.0 buffer. The ionic strength at pH 7.0 is comparable to that of SmDHFR at pH 5.0. SmDHFR is dihydrofolate reductase, and (-30)sfGFP is a superfolded green fluorescent protein with a negative charge of 30. DETAILED DESCRIPTION
[0037] The present invention will be further described below in conjunction with specific embodiments, and the above and / or other advantages of the present invention will become more apparent.
[0038] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.
[0039] In the following examples, the yield of tetrahydrofolate was determined by HPLC. Specific measurement parameters were as follows: Tetrahydrofolate concentration was determined using an Agilent HPLC 1260 system, with a mobile phase consisting of 84% 20 mM potassium phosphate (pH 7.2) and 16% methanol, passed through a ValueLab 5LC C18 column (250 x 4.6 mM; particle size 5 μm). Detection was performed with a UV detector set to a wavelength of 290 nm. The column temperature was maintained at 40°C, the injection volume was 10 μL, and the flow rate was 0.8-1 mL / min. A standard curve was prepared to quantitatively determine the concentration of tetrahydrofolate in the sample.
[0040] Example 1: Construction of recombinant plasmids pRSFDuet-SpyCatcher-folA, pRSFDuet-fdh and pRSFDuet-sfGFP-SpyTag
[0041] 1. Construction of recombinant plasmids pRSFDuet-SpyCatcher-folA and pRSFDuet-fdh
[0042] (1) Amplification of target gene
[0043] The target genes are the dihydrofolate reductase SmDHFR gene folA, the formate dehydrogenase pseFDH gene fdh, and the SpyCatcher protein gene. The dihydrofolate reductase SmDHFR is derived from Serratia marcescens, and its amino acid sequence is shown in SEQ ID NO.1, and the corresponding nucleotide sequence of the encoding gene folA is shown in SEQ ID NO.2; the formate dehydrogenase pseFDH is derived from Pseudomonas sp. 101, and its amino acid sequence is shown in SEQ ID NO.3, and the corresponding nucleotide sequence of the encoding gene fdh is shown in SEQ ID NO.4; the SpyCatcher protein has an amino acid sequence shown in SEQ ID NO.5, and the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO.6.
[0044] Through molecular biology, primers with homology arms, including SpyCatcher-F, SpyCatcher-R, folA-F, and folA-R, were designed to target the target gene. PCR amplification was performed using the folA sequence of the dihydrofolate reductase (SmDHFR) gene as a template and folA-F and folA-R as primers to obtain the folA amplification product. PCR amplification of the SpyCatcher amplification product was performed using the petduet-SpyCatcher plasmid (a laboratory-preserved plasmid) as a template and SpyCatcher-F and SpyCatcher-R as primers.
[0045] Through molecular biology, fdh-F and fdh-R were designed as target gene primers with homology arms. The fdh sequence of the artificially synthesized formate dehydrogenase pseFDH gene was used as a template for PCR amplification to obtain the fdh amplification product.
[0046] The primer sequences, PCR amplification system and amplification conditions used in the above experiments are shown in Table 1, Table 2 and Table 3 respectively.
[0047] Table 1 Target gene primer sequences
[0048]
[0049]
[0050] Table 2 PCR amplification system
[0051] sample Volume (μL) Upstream primer 1 Downstream primer 1 template 1 2xPhanta 25 <![CDATA[ddH2O]]> 22 Overall system 50
[0052] Table 3 PCR amplification conditions
[0053]
[0054] After PCR amplification is complete, pipette the sample into the wells of the prepared agarose gel for nucleic acid separation. Set the agarose gel electrophoresis instrument to 130V for 25 minutes. After the gel electrophoresis is complete, place the agarose gel in a gel imaging apparatus and observe the size of the gene fragment bands. Quickly cut the gel for the correct bright bands and recover the purified SpyCatcher, folA, and fdh fragments. Store in a -20°C freezer until ready for use.
[0055] (2) Enzyme digestion and homologous recombination
[0056] The plasmid pRSFDuet was double-digested with restriction endonucleases EcoRI and HindIII. After the digestion was completed, gel electrophoresis and gel recovery were performed to obtain a linearized pRSFDuet digestion vector. The vector was then subjected to multi-fragment homologous recombination with the SpyCatcher fragment and folA fragment purified in step (1) and transformed into Escherichia coli DH5α. After extraction and verification, the recombinant plasmid pRSFDuet-SpyCatcher-folA was obtained.
[0057] The linearized pRSFDuet-1 enzyme-cut vector and the fdh fragment purified in step (1) were homologously recombined and transformed into Escherichia coli DH5α. After extraction and verification, the recombinant plasmid pRSFDuet-fdh was obtained.
[0058] The double enzyme digestion system and homologous recombination system used in the above experimental process are shown in Tables 4 and 5.
[0059] Table 4 Double enzyme digestion system
[0060]
[0061]
[0062] Table 5 Homologous recombination system
[0063] sample Volume (μL) Linearized vector 4 Target gene 1 with homology arms 2 Target gene 2 with homology arms 2 Homologous recombinase C113 2 5× buffer enzyme 4 <![CDATA[ddH2O]]> 6 Total volume 20
[0064] 2. Construction of recombinant plasmid pRSFDuet-sfGFP-SpyTag
[0065] (-30)sfGFP, (0)sfGFP, and (+36)sfGFP represent three superfolded green fluorescent proteases (sfGFP) carrying different charges. Their amino acid sequences are shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively, and the nucleotide sequences of the corresponding encoding genes are shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively. SpyTag protein is a short peptide tag that can efficiently bind to the SpyCatcher protein in step 1 through a covalent bond to form a stable complex. The amino acid sequence of the SpyTag protein is shown in SEQ ID NO.13, and the sequence of the corresponding encoding gene is shown in SEQ ID NO.14.
[0066] The recombinant plasmid pRSFDuet-sfGFP-SpyTag is divided into three recombinant plasmids: pRSFDuet-(-30)sfGFP-SpyTag, pRSFDuet-(0)sfGFP-SpyTag, and pRSFDuet-(+36)sfGFP-SpyTag. All three recombinant plasmids were synthesized by General Biotechnology.
[0067] Example 2: Strain culture and protein purification
[0068] The recombinant plasmids pRSFDuet-SpyCatcher-folA, pRSFDuet-fdh, pRSFDuet-(-30)sfGFP-SpyTag, pRSFDuet-(0)sfGFP-SpyTag, and pRSFDuet-(+36)sfGFP-SpyTag obtained in Example 1 were chemically transformed into Escherichia coli BL21 (DE3) to obtain recombinant Escherichia coli BL21-SpyCatcher-folA, BL21-fdh, BL21-(-30)sfGFP-SpyTag, BL21-(0)sfGFP-SpyTag, and BL21-(+36)sfGFP-SpyTag.
[0069] The obtained recombinant E. coli were cultured in 150 mL of TB liquid medium at 37 °C until OD 600 When the pH reaches 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.75 mM to the culture medium, set the shaker temperature to 18°C, and continue culturing for 14-16 hours to induce protein expression. After induction, collect the cells by centrifugation at 6000 rpm and 4°C for 8 minutes, wash the cells twice with 50 mM phosphate buffer (pH 7.0), collect the cells and resuspend them in phosphate buffer. Lyse the cells by ultrasonication on ice, centrifuge at 4000 rpm for 15 minutes at 4°C, separate the supernatant and precipitate, and the resulting supernatant is the crude enzyme solution.
[0070] Using Ni produced by SUNRESIN Soluble protein was purified by FF (IDA) affinity chromatography column. The crude enzyme solution was mixed with Ni for 30 min, and nonspecific proteins were eluted using a low concentration of imidazole (50 mM phosphate buffer containing 300 mM NaCl, imidazole concentration was 30 mM). The target protein was then eluted and collected using a high concentration of imidazole (50 mM phosphate buffer containing 300 mM NaCl, imidazole concentration was 300 mM). The target protein was concentrated, and the buffer was replaced with a storage buffer (50 mM phosphoric acid, pH 7.0) using a centrifugal filter device produced by Milliproe to obtain each pure enzyme solution. Protein concentration was determined using a protein assay kit produced by Tiangen Biotech.
[0071] The pure enzyme solution of the fused dihydrofolate reductase SpyCatcher-SmDHFR was mixed with the pure enzyme solution of different fused superfolded green fluorescent protease sfGFP (+36sfGFP-SpyTag, 0sfGFP-SpyTag, -30sfGFP-SpyTag) at equimolar concentrations at 4°C for 8-12 hours to obtain the pure enzyme solution of the fused superfolded green fluorescent protein-fused dihydrofolate reductase +36sfGFP-6GS-SmDHFR, 0sfGFP-6GS-SmDHFR, and -30sfGFP-6GS-SmDHFR covalently linked by SpyCatcher / SpyTag. The purity of the isolated protein was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using the Bio-Rad Mini-Protean Tetra electrophoresis system produced by Bio-Rad. The prepared pure enzyme solution was further verified by gel electrophoresis, as shown in FIG. Figure 1 As shown, the protein size of SmDHFR is 19.9 kDa, the protein size of SpyCatcher-SmDHFR (i.e., SpyCatcher Sm in the figure) is 33.1 kDa, the protein sizes of +36sfGFP SpyTag, 0sfGFPSpyTag, and -30sfGFP SpyTag are all 33.6 kDa, and the protein sizes of +36sfGFP-6GS-SmDHFR, 0sfGFP-6GS-SmDHFR, and -30sfGFP-6GS-SmDHFR are all 66.7 kDa.
[0072] Example 3 Synthesis of Tetrahydrofolic Acid by In Vitro Enzymatic Reaction
[0073] In order to evaluate the effects of different superfolded green fluorescent proteins (sfGFP) on the catalytic activity of dihydrofolate reductase at different pH levels (5.0, 6.0, and 7.0), appropriate amounts of pure enzyme solution were used in in vitro enzyme catalysis experiments, and the following control experiments were set up.
[0074] The total volume of the reaction system was 1 mL, including 20 mM folic acid, 60 mM sodium formate, 10 mM NADP + 15 control experiments were performed using 10 μM pseFDH pure enzyme solution, 100 μM (SmDHFR, SpyCatcher-SmDHFR, +36sfGFP-6GS-SmDHFR, 0sfGFP-6GS-SmDHFR, or -30sfGFP-6GS-SmDHFR) pure enzyme solution, and 200 mM pH 5.0 acetate buffer (or pH 6.0 or pH 7.0 phosphate buffer). All catalytic reactions were performed at 35-40°C in an anaerobic chamber for 2 hours.
[0075] The results are as follows Figure 2 As shown in Figure a, fusion with SpyCatcher enhances the catalytic activity of dihydrofolate reductase under all pH conditions, and the yield is further improved after covalent linkage with (-30)sfGFP. Covalent linkage with (0)sfGFP does not significantly improve the yield, and covalent linkage with (+36)sfGFP is inhibited. It is worth noting that the tetrahydrofolate yield of dihydrofolate reductase covalently linked to (-30)sfGFP at pH 7.0 is comparable to that of wild-type dihydrofolate reductase at pH 5.0. These findings indicate that the fusion of dihydrofolate reductase with the superfolded green fluorescent protein (-30)sfGFP significantly affects the enzyme's microenvironment, thereby improving its apparent catalytic efficiency at neutral pH.
[0076] To further verify these results, the reaction time was further extended to determine the final tetrahydrofolate yield. Figure 2 As shown in Figure (b), the covalent complex of dihydrofolate reductase and (-30)sfGFP produced a tetrahydrofolate yield of 3270.1 μM at pH 7.0, nearly comparable to the 3448.3 μM yield of dihydrofolate reductase at pH 5.0. This result further confirms that fusion with (-30)sfGFP can enhance the catalytic efficiency of dihydrofolate reductase under neutral conditions. Furthermore, this covalent complex produced a tetrahydrofolate yield of 4223.4 μM after 17 hours of reaction at pH 5.0. Figure 3 Schematic diagrams of dihydrofolate reductase and superfolded green fluorescent protein are shown to help understand the experimental results of this example.
[0077] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.
[0078] The present invention provides a method and concept for synthesizing tetrahydrofolate based on enzyme microenvironment regulation. Numerous methods and approaches exist for implementing this technical solution. The foregoing merely represents preferred embodiments of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.
Claims
1. A method for synthesizing tetrahydrofolate based on enzyme microenvironment regulation, characterized in that: Tetrahydrofolate is synthesized using folic acid as substrate and NADPH as cofactor via the fusion of superfolded green fluorescent protein and dihydrofolate reductase. Wherein, the catalytic reaction system includes: folic acid, sodium formate, NADP + , fusion superfolded green fluorescent protein-fusion dihydrofolate reductase, formate dehydrogenase, buffer; the NADP + Reduced to NADPH by formate dehydrogenase.
2. The method according to claim 1, characterized in that The fusion superfolded green fluorescent protein-fusion dihydrofolate reductase is obtained by mixing the fusion superfolded green fluorescent protein and the fusion dihydrofolate reductase at equimolar concentrations at 4° C. for 8 to 12 hours; the amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO.
3.
3. The method according to claim 1 or 2, characterized in that The fused dihydrofolate reductase is a dihydrofolate reductase fused with a SpyCatcher protein gene, and the fused superfolded green fluorescent protein is a superfolded green fluorescent protein fused with a SpyTag protein gene.
4. The method according to claim 3, characterized in that The fusion dihydrofolate reductase is obtained by cloning a gene fragment of dihydrofolate reductase and a gene fragment of SpyCatcher protein into an expression vector, then introducing the vector into Escherichia coli to induce expression to obtain cells, and then ultrasonically disrupting and purifying the cells. The fusion superfolded green fluorescent protein is obtained by introducing a recombinant plasmid containing the gene of superfolded green fluorescent protein and the gene of SpyTag protein into Escherichia coli to induce expression to obtain cells, and then ultrasonically disrupting and purifying the cells.
5. The method according to claim 3, characterized in that The amino acid sequence of the SpyCatcher protein is shown in SEQ ID NO.5; the amino acid sequence of the dihydrofolate reductase is shown in SEQ ID NO.1; and the amino acid sequence of the SpyTag protein is shown in SEQ ID NO.
13.
6. The method according to any one of claim 3, characterized in that The superfolded green fluorescent protein includes any one of a superfolded green fluorescent protein with 30 negative charges, a superfolded green fluorescent protein without charge, and a superfolded green fluorescent protein with 36 positive charges.
7. The method according to claim 6, characterized in that The amino acid sequence of the superfolded green fluorescent protein with 30 negative charges is shown in SEQ ID NO.7; the amino acid sequence of the uncharged superfolded green fluorescent protein is shown in SEQ ID NO.8; the amino acid sequence of the superfolded green fluorescent protein with 36 positive charges is shown in SEQ ID NO.
9.
8. The method according to claim 1, characterized in that The catalytic synthesis has the following conditions: under anaerobic conditions, the reaction temperature is 35-40° C., the reaction pH is 5.0-7.0, and the reaction time is 2-17 hours.
9. The method according to claim 1, characterized in that In the catalytic reaction system, the concentration of folic acid is 0.1-50 mM, the concentration of sodium formate is 6-60 mM, and the concentration of NADP is 0.1-50 mM. + The concentration of is 1-10 mM, the concentration of fusion superfolded green fluorescent protein-fusion dihydrofolate reductase is 10-100 μM, and the concentration of formate dehydrogenase is 2-20 μM.
10. The method according to claim 1, characterized in that The buffer solution is a 50-200 mM phosphate buffer solution with a pH of 6.0-7.0 or a 50-200 mM acetate buffer solution with a pH of 5.0.