Method for efficiently preparing 2-deoxy-D-ribose

Abstract

The invention belongs to the technical field of nucleoside medicine preparation, and particularly discloses a method for efficiently preparing 2-deoxy-D-ribose. According to the method, reactants are added into Escherichia coli fermentation liquor for recombinant expression of thymidine phosphorylase, and acid hydrolysis treatment is carried out after one-step catalytic reaction to directly generate 2-deoxy-D-ribose. The method can be used for catalytically preparing the high-concentration 2-deoxy-D-ribose, so that the production of the 2-deoxy-D-ribose reaches a kilogram level, and large-scale industrialization is realized. The preparation process disclosed by the invention does not need to purify recombinase, is simple, saves cost, saves energy, reduces emission, reduces equipment investment, and further reduces production cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nucleoside drug preparation, in particular to a method for efficiently producing 2-deoxy-D-ribose. BACKGROUND

[0002] 2-Deoxy-D-ribose, also known as alpha-deoxy-D-ribonucleic acid, is formed by replacing the 2-position hydroxyl group of ribose with hydrogen, and belongs to aldopentose, also known as thymidine sugar. 2-Deoxy-D-ribose is an important component of nucleic acid, and can be combined with a base to generate nucleosides with significant antiviral and antitumor activity, so it is also a basic raw material and key intermediate for synthesizing nucleoside antiviral and antitumor drugs. At present, it is mainly used for the preparation of drugs such as zidovudine, lamivudine, stavudine and decitabine, and has good industrial application prospect.

[0003] The traditional preparation method of 2-deoxy-D-ribose is chemical synthesis, and the current more economical synthesis route in industry is a synthesis route taking glucose and cyclohexanone as starting materials, but there are still problems such as low total yield and heavy environmental pressure. In addition, with the development of economy and technology and the increasing attention of people to the living environment, more and more clean and efficient chemical production is required, and it is of great significance to develop an environmentally friendly and efficient 2-deoxy-D-ribose method with simple process and large-scale industrial production. SUMMARY

[0004] The present application provides a 2-deoxy-D-ribose preparation method with simple process, low cost and high conversion efficiency.

[0005] The present application provides a 2-deoxy-D-ribose preparation method with simple process, low cost and high conversion efficiency.

[0006] Specifically, the present application provides a method for preparing 2-deoxy-D-ribose, which is to make the fermentation broth of recombinant bacteria expressing thymidine phosphorylase into a reaction system, and then perform acid hydrolysis treatment after one-step catalytic reaction to generate 2-deoxy-D-ribose.

[0007] Preferably, the thymidine phosphorylase expressed by the recombinant bacteria has an amino acid sequence with 95% or more homology with the sequence shown in SEQ ID NO: 2.

[0008] Further preferably, the thymidine phosphorylase amino acid sequence expressed by the recombinant bacteria is shown as SEQ ID NO: 2.

[0009] Preferably, the recombinant bacteria is E. coli BL21 (DE3) transformed with the pET28a plasmid carrying the gene sequence shown as SEQ ID NO: 1.

[0010] The preparation method provided by the present application comprises the following steps: after the fermentation of the fermentation liquor of the recombinant bacteria is completed, other components of the reaction system are added.

[0011] Preferably, the other components of the reaction system comprise buffer salt and catalytic substrate, the pH of the reaction system is 4.5-5.5, and the reaction temperature is 50-55℃.

[0012] Further preferably, the buffer salt is phosphate, and the catalytic substrate is 2'-deoxythymidine.

[0013] Further preferably, the buffer salt is 50mM phosphate, and the pH is 4.5-5.5; the concentration of the catalytic substrate 2'-deoxythymidine is 300-400g / L.

[0014] Further preferably, in the reaction process, the pH of the reaction system is stabilized at 4.5-5.5 by titration of an alkali solution, and the reaction time is 18-24h.

[0015] Further preferably, in the reaction process, when the peak area of the liquid-phase detection of 2'-deoxythymidine is less than 1% after normalization, acid hydrolysis treatment is performed.

[0016] Further preferably, the acid hydrolysis treatment comprises adjusting the pH of the reaction solution to 2.5±0.1 by using an acid solution, continuing stirring for 30min-1h, and then adjusting the pH of the reaction solution to 7.0±0.1 by using an alkali solution.

[0017] Further preferably, the reaction solution after the pH adjustment by the alkali solution is filtered to obtain a clear solution containing 2-deoxy-D-ribose.

[0018] The preparation method of the present application can further comprise a purification step of the 2-deoxy-D-ribose clear solution.

[0019] Preferably, the purification step of the 2-deoxy-D-ribose clear solution comprises filtration, crystallization and drying.

[0020] Preferably, the fermentation of the recombinant bacteria comprises a rapid propagation stage and an induction recombinant protein expression stage.

[0021] Further preferably, the fermentation temperature in the rapid propagation stage is 33-37℃, and the fermentation temperature in the induction recombinant protein expression stage is 22-28℃.

[0022] More preferably, the dissolved oxygen level during the rapid reproduction stage is 25-75%; and the dissolved oxygen level during the recombinant protein expression induction stage is 25-55%.

[0023] In a further optimized formulation, glycerol is added during the recombinant protein expression induction stage.

[0024] More preferably, the added glycerol is 80% glycerol, and the addition rate is 20~30 g / L / H.

[0025] More preferably, the fermentation medium of the recombinant bacteria comprises: glycerol, peptone, yeast extract, K2HPO4·12H2O, KH2PO3, MgSO4·7H2O and trace elements.

[0026] More preferably, the trace elements include: FeCl3·6H2O, ZnSO4·7H2O, CaCl2, CuCl2 and MnSO4·H2O.

[0027] The technical solution provided by this invention is to first carry out high-density fermentation of genetically engineered bacteria. This fermentation process aims to improve the expression level and activity of recombinant enzymes and to finely control each stage of fermentation. The process includes: seed liquid preparation stage, rapid cell proliferation stage, and recombinant protein expression induction stage.

[0028] The novel method for synthesizing 2-deoxy-D-ribose of the present invention has the following beneficial effects: 1. Raw material economy: The raw materials used are inexpensive and readily available, which reduces production costs.

[0029] 2. Short synthesis route: The synthesis route is short, reducing intermediate steps and simplifying the production process.

[0030] 3. Easy to operate: Convenient to operate and easy to realize industrial production.

[0031] 4. High yield: Using 2'-deoxythymidine as raw material, the total yield is greater than 40% after two-step reaction, which improves the conversion efficiency of raw materials.

[0032] 5. Purity advantage: Almost no impurities are generated during the conversion process, which facilitates efficient subsequent purification and improves the purity of the product.

[0033] 6. High-density fermentation: Through precise control of the fermentation process, high-density fermentation is achieved, which increases the concentration of recombinant bacteria (70~100g / L on dry weight).

[0034] 7. Increased enzyme activity: It improved the enzyme activity of thymidine phosphorylase, and the reaction solution with 2-deoxy-D-ribose content exceeding 12 g / L could be obtained.

[0035] 8. Large-scale production capacity: Enables the production of 2-deoxy-D-ribose to reach kilogram-level output, achieving large-scale industrialization.

[0036] 9. Simple process: The preparation process does not require purification of recombinant enzymes. The catalytic reaction system can be prepared using fermentation broth, which is simple and saves costs.

[0037] 10. Cost savings: It greatly reduces equipment investment, and the use of small-tonnage fermenters to produce enzymes can meet the needs of large-scale industrial production of 2-deoxy-D-ribose.

[0038] 11. Energy saving and emission reduction: The process of this invention has significant energy saving and emission reduction effects, reduces the demand for water, electricity and steam, and greatly reduces the amount of waste generated, which is beneficial to environmental protection.

[0039] In summary, the novel synthesis method for 2-deoxy-D-ribose of the present invention has significant advantages in terms of raw material cost, ease of operation, yield, purity, production scale, cost savings, and environmental protection, and has a promising prospect for industrial application. Attached Figure Description

[0040] Figure 1 Example 2: SDS-PAGE electrophoresis image after IPTG-induced expression, where M represents Mark and the target protein molecular weight is 45 kDa. The samples detected from left to right are: induction cell lysate centrifuged precipitate, fermentation-terminated cell lysate, fermentation-terminated cell lysate supernatant, and fermentation-terminated cell lysate centrifuged precipitate, respectively referred to as induction precipitate, lysate, lysate supernatant, and lysate. Detailed Implementation

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] The present invention will be further described below through embodiments, but these descriptions are not intended to further limit the scope of the invention. Those skilled in the art should understand that equivalent substitutions or corresponding improvements made to the present invention still fall within the protection scope of the present invention.

[0043] Example 1: Construction and culture of genetically engineered bacteria This invention obtains the thymidine phosphorylase gene sequence from Escherichia coli using methods such as genomic database mining and homologous sequence alignment, combined with molecular biology techniques, as shown in SEQ ID NO:1, and the amino acid sequence encoding thymidine phosphorylase is shown in SEQ ID NO:2. S1: The nucleotide sequence shown in SEQ ID NO:1 was optimized according to the codon preference of E. coli to obtain the thymidine phosphorylase gene sequence, and then artificially synthesized (commissioned to Nanjing Qingke Biotechnology Co., Ltd.) to obtain the pET-28a(+)-TP recombinant plasmid. S2: The pET-28a(+)-TP recombinant plasmid carrying the TP gene was transformed into BL21(DE3) host Escherichia coli (BL21(DE3) host Escherichia coli was purchased from Nanjing Qingke Biotechnology Co., Ltd.), and plated. S3: Pick a single colony and inoculate it into an Erlenmeyer flask containing LB liquid medium (containing Kana resistance). Incubate overnight at 37°C and 180 rpm to obtain the seed culture (for glycerol preservation after verification). Then, take 2 ml of the seed culture and transfer it to an Erlenmeyer flask containing 100 ml of LB medium containing Kana resistance. Incubate at 37°C and 180 rpm for 1-2 hours. When the OD600 reaches approximately 0.6, add IPTG to a final concentration of 0.2 mM and induce at 26°C and 180 rpm for approximately 8 hours. S4: After induction, centrifuge at 10000 r / min, 4℃ for 5 min to collect bacterial cells. Resuspend the bacterial cells in a 1:10 ratio of bacterial cells to water and disrupt the cells using an ultrasonic cell disruptor. Take 5 ml of the bacterial suspension, cycle at 200 W for 5 seconds, stop for 7 seconds, and repeat 15 times on ice. S5: Centrifuge the cell lysis fluid at 4℃, 10000 r / min for 5 min, and collect the supernatant and precipitate separately. Then analyze the expression of the target protein by SDS-PAGE (see attached). Figure 1 As can be seen, the recombinant protein was mainly present in the supernatant after bacterial cell lysis. The seed culture of the strain with normal expression verification was mixed with 40% glycerol (1:1) to maintain glycerol culture and stored at -80℃.

[0044] Example 2 Fermentation by genetically engineered bacteria (1) Shake flask seed culture: The genetically engineered glycerol bacterium that has been expressed and verified was inoculated into 500 ml of liquid culture medium for activation culture: the temperature of the seed culture was 35℃ and the time was 15 h; The seed culture medium was LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH adjusted to 7.0 with 5M sodium hydroxide solution, sterilized at 121℃ for 20 min, and kanamycin was added at a concentration of 50 μg / mL before inoculation.

[0045] (2) Rapid cell propagation stage: 60L of basic culture medium was placed in a fermenter with an initial pH of 6.56. It was sterilized at 121℃ for 20 minutes, cooled to 37℃, and the pH was 6.21. The pH was adjusted to 7.0 with ammonia water. 500ml of seed inoculation solution was then introduced into the fermenter for fermentation.

[0046] The above-mentioned basic fermentation medium contains: 10 g / L glycerol, 12 g / L peptone, 24 g / L yeast extract, 12.5 g / L K₂HPO₄·3H₂O, 3.0 g / L KH₂PO₃, 0.2 g / L MgSO₄·7H₂O, trace elements at 3-5 mL / L, and kanamycin at 50 μg / mL. The trace elements are: (ferric chloride hexahydrate: 6 g / L, zinc sulfate heptahydrate: 0.58 g / L, calcium chloride: 0.2 g / L, copper chloride: 0.2 g / L, manganese sulfate monohydrate: 0.3 g / L). (3) Enzyme production stage: After 6 hours of fermentation, the OD600 of the fermentation broth was measured to be 15-20. The temperature of the fermentation broth was lowered to 26℃, and 0.1mM IPTG was added as an inducer. Fermentation continued for 22 hours, during which dissolved oxygen was maintained at 25-55%, the feeding rate was 20g / L / H, and the feed consisted of 80% glycerol. After fermentation, the OD600 of the fermentation broth was... 600 =70-80, cell dry weight is 80-100g / L.

[0047] Example 3 Catalytic production of 2-deoxy-D-ribose (100L tank) (1) Enzyme-catalyzed process: The fermentation process of the genetically engineered bacteria is the same as in Example 2. After fermentation, phosphate buffer and substrate 2'-deoxythymidine are directly added to 50 liters of fermentation broth to make the final concentration of buffer 50 mM, pH 4.5, and substrate concentration 350 g / L based on the fermentation broth. During the reaction, 10% sodium hydroxide needs to be titrated to stabilize the pH of the reaction system at 4.5, and the reaction time is 24 h. When the normalization of deoxythymidine in the liquid phase is less than 1%, acid hydrolysis reaction is carried out.

[0048] (2) Acid hydrolysis reaction: The pH of the reaction solution was adjusted to 2.5±0.1 using 1M hydrochloric acid solution, and stirring was continued for 30 min. Then, the pH of the reaction solution was adjusted to 7.0±0.1 using 10% sodium hydroxide solution. Finally, after adding flocculant, the system was filtered through a plate and frame filter press to obtain a clear liquid containing a high concentration of 2-deoxy-D-ribose.

[0049] (3) Crystallization: The supernatant was concentrated under reduced pressure at 70℃ to above 200g / L, and crystallized at 4±0.5℃ for 10h; the crystallized sample was redissolved with an appropriate amount of 50% ethanol, concentrated under reduced pressure at 45℃ to above 200g / L, and crystallized at 4±0.5℃ for 8h. Recrystallization was repeated once.

[0050] (4) Drying: The obtained crystals were dried under vacuum at 60℃ for 4-8 hours. 7.7 kg of pure 2-deoxy-D-ribose with a purity greater than 99.4% was obtained, with a total yield of 41%.

[0051] Example 4: Catalytic production of 2-deoxy-D-ribose (2-ton tank) (1) Enzyme-catalyzed process: The fermentation process of the genetically engineered bacteria is the same as in Example 2. After fermentation, phosphate buffer and substrate 2'-deoxythymidine are added to 1.4 tons of fermentation broth to make the final concentration of buffer 50 mM, pH 5.5, and substrate concentration 400 g / L based on the fermentation broth. During the reaction, 10% sodium hydroxide needs to be titrated to stabilize the pH of the reaction system to 5.5, and the reaction time is 18 h. When the deoxythymidine liquid phase normalization is less than 1%, acid hydrolysis is performed.

[0052] (2) Acid hydrolysis reaction: The pH of the reaction solution was adjusted to 2.5±0.1 using 1M hydrochloric acid solution, and stirring was continued for 1 hour. Then, the pH of the reaction solution was adjusted to 7.0±0.1 using 10% sodium hydroxide solution. Finally, after adding flocculant, the system was filtered through a plate and frame filter press to obtain a clear liquid containing a high concentration of 2-deoxy-D-ribose. (3) Crystallization: The supernatant was concentrated under reduced pressure at 70℃ to above 200g / L, and crystallized at 4±0.5℃ for 12h; the crystallized sample was reconstituted with an appropriate amount of 70% ethanol, concentrated under reduced pressure at 45±0.5℃ to above 200g / L, and crystallized at 4±0.5℃ for 10h. Recrystallization was repeated once.

[0053] (4) Drying: The obtained crystals were dried under vacuum at 60℃ for 4-8 hours. 240.8 kg of pure 2-deoxy-D-ribose with a purity greater than 99.6% was obtained, with a total yield of 43%.

Claims

1. A method for producing 2-deoxy-D-ribose, characterized by, The fermentation broth of the recombinant bacteria expressing thymidine phosphorylase is prepared into a reaction system, and after one-step catalytic reaction, acid hydrolysis treatment is performed to generate 2-deoxy-D-ribose.

2. The production method according to claim 1, characterized by, The thymidine phosphorylase expressed by the recombinant bacteria has an amino acid sequence that is 95% or more homologous to the sequence shown in SEQ ID NO:

2.

3. The production method according to claim 2, characterized by, The thymidine phosphorylase expressed by the recombinant bacteria has an amino acid sequence shown in SEQ ID NO:

2.

4. The production method according to claim 3, characterized by, The recombinant bacteria are Escherichia coli BL21 (DE3) transformed with a pET28a plasmid carrying the gene sequence shown in SEQ ID NO:

1.

5. The production method according to any one of claims 1 to 4, characterized by, The fermentation broth of the recombinant bacteria is added with other components of the reaction system after fermentation is completed.

6. The production method according to claim 5, wherein The other components of the reaction system include buffer salt and catalytic substrate, the pH value of the reaction system is 4.5 to 5.5, and the reaction temperature is 35°C to 40°C.

7. The production method according to claim 6, wherein The buffer salt is phosphate, and the catalytic substrate is 2'-deoxythymidine.

8. The preparation method according to claim 7, characterized in that, The buffer salt is 50 mM phosphate with a pH value of 4.5 to 5.5, and the concentration of the catalytic substrate 2'-deoxythymidine is 300 g / L to 400 g / L.

9. The preparation method according to claim 6, characterized in that, During the reaction, the pH value of the reaction system is maintained at 4.5 to 5.5 by titrating alkali solution, and the reaction time is 18 hours to 24 hours.

10. The method of claim 5, wherein, During the reaction, when the liquid-phase detection peak area of 2'-deoxythymidine is normalized to less than 1%, acid hydrolysis treatment is performed.

11. The method of claim 10, wherein, The acid hydrolysis treatment includes adjusting the pH value of the reaction liquid to 2.5±0.1 with an acid solution, continuing stirring for 30 minutes to 1 hour, and then adjusting the pH value of the reaction liquid to 7.0±0.1 with an alkali solution.

12. The method of claim 11, wherein, The reaction liquid after the pH value is adjusted by the alkali solution is filtered to obtain a clear liquid containing 2-deoxy-D-ribose.

13. The method of claim 12, wherein, The preparation method further includes a step of purifying the clear liquid containing 2-deoxy-D-ribose.

14. The method of claim 13, wherein, The purification step includes filtration, crystallization, and drying.

15. The production method according to any one of claims 1 to 4, characterized by, The fermentation process of the recombinant bacteria includes a rapid propagation stage and a recombinant protein expression induction stage.

16. The method of claim 15, wherein, In the rapid propagation stage, the fermentation temperature is 33°C to 37°C, and in the recombinant protein expression induction stage, the fermentation temperature is 22°C to 28°C.

17. The preparation method according to claim 15, characterized in that, In the rapid propagation stage, the dissolved oxygen content is 25% to 75%, and in the recombinant protein expression induction stage, the dissolved oxygen content is 25% to 55%.

18. The method of claim 15, wherein, In the recombinant protein expression induction stage, glycerol is supplemented.

19. The production method according to any one of claims 1 to 4, characterized by, The fermentation medium of the recombinant bacteria includes glycerol, proteose peptone, yeast extract, K2HPO4·12H2O, KH2PO4, MgSO4·7H2O, and trace elements.

20. The method of claim 19, wherein, The trace elements include FeCl3·6H2O, ZnSO4·7H2O, CaCl2, CuCl2, and MnSO4·H2O.

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