A one-pot multi-enzyme method for synthesizing pseudouridine
The synthesis of pseudouridine by one pot multi-enzyme method has solved the problems of cumbersome synthesis steps, low yields and complex biocatalytic solutions in the existing technology, and achieved low-cost and high-efficiency pseudouridine synthesis, which is suitable for large-scale industrial production and environmental protection.
Patent Information
- Application Number
- CN202411920269.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-25
- Publication Date
- 2025-04-08
AI Technical Summary
In the prior art, the preparation method of pseudouridine has problems such as cumbersome synthesis steps, low yield, high safety risks, low yield of biocatalytic schemes, high raw material costs and complex operating steps, and it is difficult to apply to large-scale industrial production.
Pseudouridine was synthesized by one pot multi-enzyme method. By weighing D-ribose, ATP, metal cofactors, uracil and other reactants, the pH was adjusted to 7.0-8.0, ribokinase, pseudouridine-5-phosphoglycosidase and 5'-nucleotide enzyme were added, and the reaction was purified to obtain pseudouridine.
This method greatly shortens the reaction process and time, has low raw material costs, mild reaction conditions, high yield and stable output. It is suitable for large-scale industrial production and is environmentally friendly.
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Figure CN119776462A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomedicine, and in particular to a method for synthesizing pseudouridine by a one-pot multi-enzyme method. Background Art
[0002] Pseudouridine, known as the "fifth nucleoside" of RNA, is the most abundant modified nucleoside on RNA. Studies have revealed that the introduction of pseudouridine into RNA can significantly reduce its immunogenicity, and this reduction effect is positively correlated with the introduction ratio of pseudouridine. Further studies have found that when pseudouridine is completely replaced by uridine, the immunogenicity of mRNA is greatly reduced, while its stability is enhanced and its translation ability is also improved.
[0003] However, the preparation of pseudouridine currently relies mainly on chemical synthesis methods, but this method has many challenges, such as cumbersome synthesis steps, low yield, and the need to use a large amount of flammable and explosive reagents during the synthesis process, which increases the safety risk of production. In recent years, although the biocatalytic synthesis of pseudouridine has gradually emerged, the existing biocatalytic scheme also faces problems such as low yield, high raw material cost, and complex operation steps, making it difficult to truly apply to large-scale industrial production.
[0004] Therefore, seeking and exploring a low-cost, high-efficiency method for synthesizing pseudouridine has become an important issue that needs to be urgently addressed in this field. Summary of the invention
[0005] Based on the above description, the present invention provides a one-pot multi-enzyme method for synthesizing pseudouridine, so as to provide a low-cost and high-efficiency method for synthesizing pseudouridine.
[0006] The technical solution of the present invention to solve the above technical problems is as follows: The present invention provides a method for synthesizing pseudouridine by a one-pot multi-enzyme method, comprising the following steps: S1: Weigh D-ribose, ATP and metal cofactor Mn 2+ Mg 2+ and K + salt, and dissolve it in deionized water to obtain a first solution; then weigh uracil, and dissolve it in deionized water to obtain a second solution; S2: mixing the first solution and the second solution, stirring and mixing them evenly, adjusting the pH to 7.0-8.0, and setting aside; S3: adding ribokinase, pseudouridine-5-phosphatase, and 5'-nucleotidase to react fully to obtain a pseudouridine reaction solution; S4: Purify the pseudouridine reaction solution, filter and dry to obtain pseudouridine.
[0007] Based on the above technical solution, the present invention can also be improved as follows.
[0008] Furthermore, the amino acid sequences of ribokinase, pseudouridine-5-phosphatase, and 5'-nucleotidase selected in step S3 are: The amino acid sequence of the ribokinase is shown in SEQ ID NO: 1; The amino acid sequence of the pseudouridine-5-phosphatase is shown in SEQ ID NO: 2; The amino acid sequence of the 5'-nucleotidase is shown in SEQ ID NO:3.
[0009] Furthermore, the reaction time of step S3 is 2-4 hours, the reaction temperature is 37±1° C., and the pH of the reaction system is 7.0-8.0.
[0010] Furthermore, the metal ion Mn in step S1 2+ The salt is MnCl2; Metal ion Mg 2+ The salt is MgCl2; Metal ion K + The salt was KCl.
[0011] Furthermore, in step S1, the concentrations of the reactants are: The concentrations of D-ribose and ATP were both 50 mM; The concentrations of MnCl2 salt and MgCl2 salt were 10 mM respectively; the concentration of KCl salt was 100 mM; The concentration of uracil was 50 mM.
[0012] Further, in step S3, the final concentration of ribokinase is 0.5 mg / mL; The final concentration of pseudouridine-5-phosphatase was 0.5 mg / mL; The final concentration of 5'-nucleotidase was 0.1 mg / mL.
[0013] Furthermore, before step S1, the process also includes: construction and verification of a vector and expression and purification of an enzyme.
[0014] Furthermore, the construction and verification of the vector specifically includes: The selected ribokinase, pseudouridine-5-phosphatase, and 5'-nucleotidase amino acid sequences were connected to the vector pET-28a by seamless cloning; The NdeI and XhoI restriction sites were used at both ends of the connected amino acid sequence to digest the target gene and pET-28a plasmid, and the target gene fragment and vector fragment were recovered by agarose gel electrophoresis; Use T4 DNA ligase to carry out ligation reaction, transform competent E. coli DH5α, and spread on plates with kanamycin; After picking a single clone for colony PCR verification, select the successfully connected strain to extract the plasmid for sequencing to verify the accuracy of the target fragment sequence.
[0015] Furthermore, the expression and purification of the enzyme specifically includes: After the correctly sequenced plasmid was transformed into the BL21 (DE3) strain, a single clone was picked and cultured in an LB tube overnight; The next day, 200 mL of LB medium was inoculated at a ratio of 1:100, cultured at 37°C for 6 h, and inoculated into a 5 L fermenter; When the optical density was close to 30 at 37°C, the temperature was lowered to 20°C, 0.2 mM IPTG was added, and the cells were induced for 12 h before being removed from the pot. After the bacteria were collected by centrifugation, they were resuspended in a buffer solution, and the recombinant protein was released by high-pressure crushing. The target protein was then purified by nickel column affinity chromatography.
[0016] Furthermore, step S4 specifically includes: S401, ultrafiltration to remove protein: the reaction solution is filtered using a 5 μm filter membrane under positive pressure, and the protein in the reaction solution is removed by ultrafiltration using a 5 kDa ultrafiltration membrane; S402, barium salt precipitation and nanofiltration: add 1M BaCl2 solution, stir well and centrifuge, repeat the operation until no white precipitate is generated in the supernatant; add 2M Na2SO4 solution, stir well and filter, repeat the operation until no white precipitate is generated in the supernatant; use 200Da filter membrane to nanofilter the supernatant, and collect the nanofiltration permeate; S403, electrodialysis desalination: desalting the nanofiltration permeate using an electrodialysis device until the conductivity drops below 100 μS / cm; S404, concentrating the reaction solution after desalting, precipitating solid, filtering and drying to obtain pseudouridine.
[0017] Compared with the prior art, the technical solution of this application has the following beneficial technical effects: The one-pot multi-enzyme method for synthesizing pseudouridine provided by the present invention has the following beneficial effects compared with the existing methods: The method for synthesizing pseudouridine uses a "one-pot multi-enzyme" method to synthesize pseudouridine. First, D-ribose and ATP generate ribose-5-phosphate under the action of ribokinase, and then react with uracil under the catalysis of pseudouridine-5-phosphate glycosidase to generate pseudouridine monophosphate (reversible reaction). Finally, pseudouridine is generated under the action of 5'-nucleotidase, and the reversible reaction is driven toward the direction of generating pseudouridine monophosphate. The three-step reaction can be carried out in one pot to directly obtain the product pseudouridine.
[0018] This method greatly shortens the reaction process and reaction time, and the raw materials required are inexpensive, the reaction process is simple and rapid, and no expensive and complex instruments and equipment are required. The reaction conditions are mild, the yield is high and the output is stable. The use of enzymes is also environmentally friendly. It is a green and efficient pseudouridine synthesis technology that can be used on an industrial scale. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0020] Figure 1 A schematic diagram of a one-pot multi-enzyme method for synthesizing pseudouridine provided in an embodiment of the present invention; Figure 2 A schematic diagram of the synthesis principle of the method for synthesizing pseudouridine by a one-pot multi-enzyme method provided in an embodiment of the present invention; Figure 3 The HPLC test results of the lower tank reaction liquid provided in the embodiment of the present invention; Figure 4 The HPLC test results of pseudouridine provided in the embodiments of the present invention; Figure 5 The figure is the HPLC test result of the pseudouridine standard of the present invention. DETAILED DESCRIPTION
[0021] The specific implementation of the present invention is described in detail below. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the protection scope of the present invention.
[0022] The endpoints and any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.
[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
[0024] In the present invention, the conventional reagents and raw materials used in the experiments are all commercially available.
[0025] Combined with Figure 1 and Figure 2The one-pot multi-enzyme method for synthesizing pseudouridine provided by the present invention is as follows: Figure 1 As shown, the following steps are included: Step S1: Weigh D-ribose, ATP and metal cofactor Mn 2+ Mg 2+ and K + The salt is dissolved in deionized water to obtain a first solution; then uracil is weighed and dissolved in deionized water to obtain a second solution.
[0026] Among them, the metal ion Mn 2+ The salt is preferably MnCl2; Metal ion Mg 2+ The salt is preferably MgCl2; Metal ion K + The salt is preferably KCl.
[0027] Correspondingly, the concentrations of the reactants are: The concentrations of D-ribose and ATP were both 50 mM; The concentrations of MnCl2 salt and MgCl2 salt were 10 mM respectively; the concentration of KCl salt was 100 mM; The concentration of uracil was 50 mM.
[0028] During the above dissolution process, the solution may be heated to 80°C to assist dissolution.
[0029] In addition, in an optional embodiment, before this step, it also includes: construction and verification of the vector and expression and purification of the enzyme.
[0030] The construction and verification of the vector specifically includes: The selected ribokinase, pseudouridine-5-phosphatase, and 5'-nucleotidase amino acid sequences were connected to the vector pET-28a by seamless cloning; The NdeI and XhoI restriction sites were used at both ends of the connected amino acid sequence to digest the target gene and pET-28a plasmid, and the target gene fragment and vector fragment were recovered by agarose gel electrophoresis; Use T4 DNA ligase to carry out ligation reaction, transform competent E. coli DH5α, and spread on plates with kanamycin; After picking a single clone for colony PCR verification, select the successfully connected strain to extract the plasmid for sequencing to verify the accuracy of the target fragment sequence.
[0031] Enzyme expression and purification specifically include: After the correctly sequenced plasmid was transformed into the BL21 (DE3) strain, a single clone was picked and cultured in an LB tube overnight; The next day, 200 mL of LB medium was inoculated at a ratio of 1:100, cultured at 37°C for 6 h, and inoculated into a 5 L fermenter; When the optical density was close to 30 at 37°C, the temperature was lowered to 20°C, 0.2 mM IPTG was added, and the cells were induced for 12 h before being removed from the pot. After the bacteria were collected by centrifugation, they were resuspended in a buffer solution, and the recombinant protein was released by high-pressure crushing. The target protein was then purified by nickel column affinity chromatography.
[0032] Step S2: Mix the first solution and the second solution and stir them to mix them evenly, and adjust the pH to 7.0-8.0 for later use, wherein the pH is preferably 7.5.
[0033] Step S3: adding ribokinase, pseudouridine-5-phosphatase, and 5'-nucleotidase, and reacting sufficiently to obtain a pseudouridine reaction solution.
[0034] Specifically, in this step, the reaction time is 2-4 hours, the reaction temperature is 37±1° C., and the pH of the reaction system is 7.0-8.0, preferably 7.5.
[0035] Among them, the amino acid sequences of the selected ribokinase, pseudouridine-5-phosphatase, and 5'-nucleotidase are: The amino acid sequence of ribokinase is shown in SEQ ID NO: 1; SEQ ID NO: 1 MQNAGSLVVLGSINADHILNLQSFPTPGETVTGNHYQVAFGGKGANQAVAAGRSGANIAFIACTGDDSIGESVRQQLATDNIDITPVSVIKGESTGVALIFVNGEGENVIGIHAGANAALSPALVEAQRERIANASALLMQLESPLESVMAAAK IAHQNKTIVALNPAPARELPDELLALVDIITPNETEAEKLTGIRVENDEDAAKAAQVLHEKGIRTVLITLGSRGVWASVNGEGQRVPGFRVQAVDTIAAGDTFNGALITALLEEKPLPEAIRFAHAAAAIAVTRKGAQPSVPWREEIDAFLDRQR The amino acid sequence of pseudouridine-5-phosphatase is shown in SEQ ID NO: 2; SEQ ID NO: 2 MKTKYLVESCLLTHGLRSITDGEVIRAWDGVNATFAWIDEGKIQTGCMERFIEFRKRKIKLRVNCKNMDEMLKKRVPGALTAAGTMEACHRLGIPVTVTCGIGGIGNIPGETICSDLPALKNIPV NLVATSPKDMIDVGQTFLWLRERGVKILGYHTDYCTGYVFESMHEKLDGMFETVPFQDKGKKLLLNPIPIEKRIHDNGILEKAVKEAVEAVKKGEYFHPTVNGALDRLTNGRTSEIQLESIVENA KLAGYIAEFK The amino acid sequence of 5'-nucleotidase is shown in SEQ ID NO: 3; SEQ ID NO: 3 MKWDWIFFDADETLTFDSFTGLQRMFLDYSVTFTAEDFQDYQAVNKPLWVDYQNGAITSLQLQHGRFESWAERLNVEPGKLNEAFINAMAEICTPLPGAVSLLNAIRGNAKI GIITNGFSALQQVRLERTGLRDYFDLLVISEEVGVAKPNKKIFDYALEQAGNPDRSRVLMVGDTAESDILGGINAGLATCWLNAHHREQPEGIAPTWTVSSLHELEQLLCKH.
[0036] Correspondingly, the final concentration of ribokinase is preferably 0.5 mg / mL; The final concentration of pseudouridine-5-phosphatase is preferably 0.5 mg / mL; The final concentration of 5'-nucleotidase is preferably 0.1 mg / mL.
[0037] Step S4: purifying the pseudouridine reaction solution, filtering and drying to obtain pseudouridine.
[0038] Specifically include: Step S401, ultrafiltration to remove protein: the reaction solution is filtered using a 5 μm filter membrane under positive pressure, and a 5 kDa ultrafiltration membrane is used to remove protein in the reaction solution; Step S402, barium salt precipitation and nanofiltration: add 1M BaCl2 solution, stir well, and then centrifuge, repeat the operation until no white precipitate is generated in the supernatant; add 2M Na2SO4 solution, stir well, and then filter, repeat the operation until no white precipitate is generated in the supernatant; use a 200Da filter membrane to nanofilter the supernatant, and collect the nanofiltration permeate; Step S403, electrodialysis desalination: desalting the nanofiltration permeate using an electrodialysis device until the conductivity drops below 100 μS / cm; Step S404, concentrating the reaction solution after desalting to precipitate a large amount of solid, filtering and drying to obtain pseudouridine.
[0039] Correspondingly, the synthetic idea of the above-mentioned one-pot multi-enzyme method for synthesizing pseudouridine is as follows Figure 2 shown.
[0040] The following is combined with Figures 3 to 5 , further provide specific embodiments for introduction: The one-pot multi-enzyme method for synthesizing pseudouridine provided in this embodiment comprises the following specific steps: 1. Expression and purification of ribokinase (Rbsk) (1) Ribokinase is derived from Escherichia coli Escherichia coli , synthesize ribokinase optimized for Escherichia coli codons, with NdeI and XhoI restriction sites at both ends respectively; use restriction endonucleases NdeI and XhoI to digest the target gene and pET-28a plasmid, recover the target gene fragment and vector fragment by agarose gel electrophoresis, use T4 DNA ligase for ligation reaction, transform Escherichia coli DH5α competent cells, and coat plates with kanamycin; pick single clones for colony PCR verification, select the successfully connected strains to extract plasmids for sequencing, and verify the accuracy of the target fragment sequence.
[0041] (2) After the correctly sequenced plasmid was transformed into the BL21 (DE3) strain, a single clone was picked and cultured overnight in an LB test tube; the next day, 200 mL of LB medium was inoculated at a ratio of 1:100, cultured at 37°C for 6 h, and then inoculated into a 5 L fermenter; when the OD was close to 30 at 37°C, the temperature was lowered to 20°C, 0.2 mM IPTG was added, and the culture was induced for 12 h before being removed from the fermenter; the cells were collected by centrifugation at 7000 g and resuspended in buffer, and the recombinant protein was released by high-pressure crushing, and then purified by nickel column affinity chromatography to obtain the target protein.
[0042] 2. Expression and purification of pseudouridine-5-phosphatase (YeiN) (1) Pseudocouridine 5-phosphatase is derived from microbial strains Dorea formicigenernsATCC 27755, synthesize pseudouridine-5-phosphatase optimized for Escherichia coli codons, with NdeI and XhoI restriction sites at both ends respectively; use restriction endonucleases NdeI and XhoI to digest the target gene and pET-28a plasmid, recover the target gene fragment and vector fragment by agarose gel electrophoresis, use T4 DNA ligase for ligation reaction, transform Escherichia coli DH5α competent cells, and coat the plates with kanamycin; pick single clones for colony PCR verification, select the successfully connected strains to extract plasmid sequencing, and verify the accuracy of the target fragment sequence.
[0043] (2) After the correctly sequenced plasmid is transformed into the BL21 (DE3) strain, a single clone is picked and cultured overnight in an LB test tube. The next day, 200 mL of LB medium is inoculated at a ratio of 1:100, cultured at 37°C for 6 h, and inoculated into a 5L fermenter. When the OD is close to 30 at 37°C, the temperature is lowered to 20°C, 0.2 mM IPTG is added, and the fermenter is removed after induction for 12 h; the cells are collected by centrifugation at 7000 g and resuspended in buffer. After high-pressure crushing to release the recombinant protein, the target protein is purified by nickel column affinity chromatography.
[0044] 3. Expression and purification of 5'-nucleotidase (YjjG) (1) 5'-nucleotidase from Escherichia coli Escherichia coli , synthesize 5'-nucleotidase optimized for Escherichia coli codons, with NdeI and XhoI restriction sites at both ends respectively; use restriction endonucleases NdeI and XhoI to digest the target gene and pET-28a plasmid, recover the target gene fragment and vector fragment by agarose gel electrophoresis, use T4 DNA ligase for ligation reaction, transform Escherichia coli DH5α competent cells, and coat plates with kanamycin; pick single clones for colony PCR verification, select the successfully connected strains to extract plasmids for sequencing, and verify the accuracy of the target fragment sequence.
[0045] (2) After the correctly sequenced plasmid was transformed into the BL21 (DE3) strain, a single clone was picked and cultured overnight in an LB test tube; the next day, 200 mL of LB medium was inoculated at a ratio of 1:100, cultured at 37°C for 6 h, and then inoculated into a 5 L fermenter; when the OD was close to 30 at 37°C, the temperature was lowered to 20°C, 0.2 mM IPTG was added, and the culture was induced for 12 h before being removed from the fermenter; the cells were collected by centrifugation at 7000 g and resuspended in buffer, and the recombinant protein was released by high-pressure crushing, and then purified by nickel column affinity chromatography to obtain the target protein.
[0046] 4. One-pot multi-enzyme synthesis of pseudouridine (1) Weigh 11.51 g (50 mM) D-ribose, 27.56 g (50 mM) ATP·2Na, 1.26 g (10 mM) MnCl2, 2.03 g (10 mM) MgCl2·6H2O, and 7.46 g (100 mM) KCl, and dissolve them in 300 ml of deionized water. Then weigh 5.60 g (50 mM) uracil and dissolve it in 300 ml of deionized water, which can be heated to 80°C to aid dissolution.
[0047] (2) Pour the two prepared solutions into a 2L reactor and set the stirring speed to 150 rpm, the temperature to 37°C, and the pH to 7.5. When the temperature rises to 37°C and the pH reaches 7.5 (±0.05), add 0.5 mg / ml ribokinase, 0.5 mg / ml pseudouridine-5-phosphatase, and 0.1 mg / ml 5'-nucleotidase to a final concentration of 1L. Use 2M NaOH solution to automatically adjust the pH. After 2 hours, take 500 μl of the reaction solution every 30 minutes, heat it in a boiling water bath for 10 minutes, centrifuge it, and take the supernatant for HPLC detection. Terminate the reaction when the pseudouridine production stabilizes.
[0048] The reaction solution test results are as follows Figure 3 shown.
[0049] 5. Purification of pseudouridine reaction solution (1) Ultrafiltration to remove proteins: The reaction solution was filtered using a 5 μm filter membrane under positive pressure, and then ultrafiltration was performed using a 5 kDa ultrafiltration membrane to remove proteins from the reaction solution.
[0050] (2) Barium salt precipitation and nanofiltration: Add an appropriate amount of 1M BaCl2 solution, stir thoroughly, and then centrifuge. Repeat the operation until no white precipitate is formed in the supernatant. Then add 2M Na2SO4 solution, stir thoroughly, and then filter. Repeat the operation until no white precipitate is formed in the supernatant. Use a 200Da filter membrane to nanofilter the supernatant and collect the permeate.
[0051] (3) Electrodialysis desalination: The nanofiltration permeate is desalted using an electrodialysis device until the conductivity drops below 100 μS / cm.
[0052] (4) After desalting, the reaction solution is concentrated to a smaller volume. A large amount of product will precipitate, which is filtered and dried to obtain the pseudouridine product.
[0053] 6. HPLC (High Performance Liquid Chromatography) Detection of Pseudouridine Commercially available pseudouridine (Sigma-Aldrich, catalog number SMB00912) was used as the pseudouridine standard. The HPLC results of the pseudouridine standard are as follows: Figure 5 shown.
[0054] Column: Waters XBridge BEH Amide, 250 × 4.6 mm, 5 μm; Wavelength: 262nm; Column temperature: 40°C; Flow rate: 1 mL / min; Mobile phase A: 0.2% KH2PO4 aqueous solution (2 g KH2PO4 was weighed and dissolved in 1 L water); Mobile phase B: ACN.
[0055] Elution gradient:
[0056] The pseudouridine HPLC test results obtained are as follows Figure 4 shown.
[0057] In this embodiment, the one-pot multi-enzyme method was used to obtain a pseudouridine yield of 12 g / L, a conversion rate of 99%, and a purity of 99.7%.
[0058] Comparative Example 1 This comparative example is a prior research technology of the applicant of the present invention, in which ribose-5-phosphate and pseudouridine-5-phosphatase mutant are used to synthesize pseudouridine monophosphate, and then pseudouridine is obtained after dephosphorylation and separation. That is, only ribokinase and pseudouridine-5-phosphatase are used.
[0059] The pseudouridine yield obtained by using the pseudouridine-5-phosphatase mutant was 7.9 g / L, the conversion rate was 51%, and the purity was 98.3%.
[0060] Comparative Example 2 The difference between this comparative example and the above-mentioned example is that only ribokinase and 5'-nucleotidase are used in the "one-pot multi-enzyme synthesis of pseudouridine".
[0061] Pseudouridine cannot be obtained by this method.
[0062] Comparative Example 3 The difference between this comparative example and the above-mentioned example is that only pseudouridine-5-phosphatase and 5'-nucleotidase are used in the "one-pot multi-enzyme synthesis of pseudouridine".
[0063] Pseudouridine cannot be obtained by this method.
[0064] In summary, in the one-pot multi-enzyme method for synthesizing pseudouridine provided by the embodiment of the present invention, D-ribose and ATP first generate ribose-5-phosphate under the action of ribokinase, and then generate pseudouridine monophosphate (reversible reaction) with uracil under the catalysis of pseudouridine-5-phosphoglycosidase, and finally generate pseudouridine under the action of 5'-nucleotidase, pulling the reversible reaction towards the direction of generating pseudouridine monophosphate. The three-step reaction can be carried out in one pot to directly obtain the product pseudouridine. This method greatly shortens the reaction process and reaction time, and the raw materials required are cheap, the reaction process is simple and rapid, no expensive and complex instruments and equipment are required, the reaction conditions are mild, the yield is high and the output is stable, and the use of enzyme method is also environmentally friendly. It is a green and efficient pseudouridine synthesis technology that can be used for industrial large-scale.
[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A one-pot multi-enzyme method for synthesizing pseudouridine, characterized in that: The steps include: S1: Weigh D-ribose, ATP and metal cofactor Mn 2+ Mg 2+ and K + The salt is dissolved in deionized water to obtain a first solution; Then weigh uracil, dissolve it in deionized water, and obtain a second solution; S2: mixing the first solution and the second solution and stirring them uniformly, adjusting the pH to 7.0-8.0, and setting aside; S3: adding ribokinase, pseudouridine-5-phosphatase, and 5'-nucleotidase to react fully to obtain a pseudouridine reaction solution; S4: Purify the pseudouridine reaction solution, filter and dry to obtain pseudouridine.
2. The one-pot multi-enzyme method for synthesizing pseudouridine according to claim 1, characterized in that: The amino acid sequences of ribokinase, pseudouridine-5-phosphatase, and 5'-nucleotidase selected in step S3 are: The amino acid sequence of the ribokinase is shown in SEQ ID NO: 1; The amino acid sequence of the pseudouridine-5-phosphatase is shown in SEQ ID NO: 2; The amino acid sequence of the 5'-nucleotidase is shown in SEQ ID NO:
3.
3. The method for synthesizing pseudouridine by one-pot multi-enzyme method according to claim 2, characterized in that: The reaction time of step S3 is 2-4h, the reaction temperature is 37±1°C, and the pH of the reaction system is 7.0-8.
0.
4. The method for synthesizing pseudouridine by one-pot multi-enzyme method according to claim 2, characterized in that: The metal ion Mn in step S1 2+ The salt is MnCl2; Metal ion Mg 2+ The salt is MgCl2; Metal ion K + The salt was KCl.
5. The one-pot multi-enzyme method for synthesizing pseudouridine according to claim 4, characterized in that: In step S1, the concentrations of the reactants are: The concentrations of D-ribose and ATP were both 50 mM; The concentrations of MnCl2 salt and MgCl2 salt were 10 mM respectively; the concentration of KCl salt was 100 mM; The concentration of uracil was 50 mM.
6. The one-pot multi-enzyme method for synthesizing pseudouridine according to claim 1, characterized in that: In step S3, the final concentration of ribokinase is 0.5 mg / mL; The final concentration of pseudouridine-5-phosphatase was 0.5 mg / mL; The final concentration of 5'-nucleotidase was 0.1 mg / mL.
7. The method for synthesizing pseudouridine by one-pot multi-enzyme method according to claim 2, characterized in that: Before step S1, the process also includes: construction and verification of the vector and expression and purification of the enzyme.
8. The method for synthesizing pseudouridine by one-pot multi-enzyme method according to claim 7, characterized in that: The construction and verification of the vector specifically include: The selected ribokinase, pseudouridine-5-phosphatase, and 5'-nucleotidase amino acid sequences were connected to the vector pET-28a by seamless cloning; The NdeI and XhoI restriction sites were used at both ends of the connected amino acid sequence to digest the target gene and pET-28a plasmid, and the target gene fragment and vector fragment were recovered by agarose gel electrophoresis; Use T4 DNA ligase to carry out ligation reaction, transform competent E. coli DH5α, and spread on plates with kanamycin; After picking a single clone for colony PCR verification, select the successfully connected strain to extract the plasmid for sequencing to verify the accuracy of the target fragment sequence.
9. The one-pot multi-enzyme method for synthesizing pseudouridine according to claim 8, characterized in that: The expression and purification of the enzyme specifically include: After the correctly sequenced plasmid was transformed into the BL21 (DE3) strain, a single clone was picked and cultured in an LB tube overnight; The next day, 200 mL of LB medium was inoculated at a ratio of 1:100, cultured at 37°C for 6 h, and inoculated into a 5 L fermenter; When the optical density was close to 30 at 37°C, the temperature was lowered to 20°C, 0.2 mM IPTG was added, and the cells were induced for 12 h before being removed from the pot. After the bacteria were collected by centrifugation, they were resuspended in a buffer solution, and the recombinant protein was released by high-pressure crushing. The target protein was then purified by nickel column affinity chromatography.
10. The one-pot multi-enzyme method for synthesizing pseudouridine according to claim 1, characterized in that: Step S4 specifically includes: S401, ultrafiltration to remove protein: the reaction solution is filtered using a 5 μm filter membrane under positive pressure, and the protein in the reaction solution is removed by ultrafiltration using a 5 kDa ultrafiltration membrane; S402, barium salt precipitation and nanofiltration: add 1M BaCl2 solution, stir well and centrifuge, repeat the operation until no white precipitate is generated in the supernatant; add 2M Na2SO4 solution, stir well and filter, repeat the operation until no white precipitate is generated in the supernatant; use 200Da filter membrane to nanofilter the supernatant, and collect the nanofiltration permeate; S403, electrodialysis desalination: desalting the nanofiltration permeate using an electrodialysis device until the conductivity drops below 100 μS / cm; S404, concentrating the reaction solution after desalting, precipitating solid, filtering and drying to obtain pseudouridine.