Method for synthesizing levofolinic acid based on enzymatic tandem reaction

Abstract

The invention relates to a method for synthesizing levofolinic acid based on enzymatic cascade reaction, and belongs to the technical field of bioengineering and pharmacy. The reaction system provided by the invention comprises dihydrofolate reductase, tetrahydrofolate formyltransferase and an NAD (P) dependent coenzyme circulation system. The method comprises the following steps: taking folic acid as a substrate, enabling the folic acid to be subjected to asymmetric hydrogenation reaction under the catalysis of dihydrofolate reductase (DHFR) to obtain an intermediate compound L-tetrahydrofolic acid, and then catalyzing the intermediate compound L-tetrahydrofolic acid by tetrahydrofolate formyltransferase (FTCD) (taking N-formyl-L-glutamic acid as a formyl donor) to obtain optically pure L-5-formyl tetrahydrofolic acid (namely levofolinate). The method for preparing the levofolinic acid does not need to separate an intermediate product, does not need to split, is high in yield, and has great application value and market prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for synthesizing levofolinate based on an enzymatic cascade reaction, belonging to the field of bioengineering and pharmaceutical technology. BACKGROUND

[0002] Folinic acid, also known as 5-formyltetrahydrofolate, can alleviate the toxic side effects of dihydrofolate reductase inhibitors such as methotrexate, and is often used as a detoxifying agent in cancer treatment. For example, it is used in combination with methotrexate, vinorelbine, and cisplatin for the treatment of advanced esophageal cancer. Folinic acid can also be used as a synergist when combined with 5-fluorouracil drugs, which can stabilize their target sites and significantly enhance anticancer efficacy. It is a core component of chemotherapy regimens for colorectal cancer. In addition, folinic acid also has the effect of stimulating white blood cell growth and can be used to treat megaloblastic anemia and leukopenia.

[0003] Folinic acid molecules contain two chiral centers, so there are multiple stereoisomers. Among these isomers, only one specific stereoisomer (corresponding to the natural L-glutamic acid configuration and a specific pteridine ring configuration) has biological activity. This active isomer can be metabolized by cells to form active forms such as 5,10-methylene tetrahydrofolate or L-5-methyl tetrahydrofolate, which directly participate in key metabolic processes such as DNA synthesis.

[0004] However, other inactive isomers (including enantiomers and diastereomers) in the mixture, which are not converted in vivo, circulate in the plasma in their original form and are slowly excreted with urine, which can lead to accumulation in the body. Preclinical studies have shown that these inactive isomers can compete with folate transport proteins on cell membranes with the active isomer. This competitive inhibition reduces the efficiency of the active isomer entering the cell, resulting in the actual therapeutic effect of the racemic mixture being lower than that of the pure active isomer at the same dose. Therefore, long-term use of racemic folinic acid may not achieve the expected therapeutic effect and may pose potential risks due to the accumulation of inactive components. Therefore, optically pure levofolinate can prevent the interference of inactive isomers in clinical use, which is particularly important for patients who receive long-term folinic acid treatment.

[0005] The existing commercially available calcium folinate product is prepared by a chemical synthesis method (US4148999; US5010194; CN114591330A; CN102399223A; J Med Chem. 1979 Jun; 22(6): 731-734.), that is, a synthetic folic acid is used as a starting material, a diastereoisomer mixture of (6S, 6R)-tetrahydrofolic acid is generated by sodium borohydride reduction, then a formylation reaction is performed, and hydrochloric acid is salified to obtain 5,10-methylene tetrahydrofolic acid hydrochloride, which is then hydrolyzed and precipitated by adding a calcium source to obtain calcium folinate composed of a diastereoisomer mixture; further, an effective resolving agent is used for crystallization resolution to prepare levofolinate (US5134235; US5391738; CN103102350A; CN101792444A; CN113679668A).

[0006] However, the method combining sodium borohydride reduction and crystallization resolution has the following disadvantages:

[0007] 1) NaBH4 has strong irritancy and is highly flammable, which has safety hazards;

[0008] 2) The product is a diastereoisomer mixture, which is difficult to separate;

[0009] 3) There are many production steps, the production yield is low, and impurities are easily left in the production process. SUMMARY

[0010] To solve the above problems, the present application provides a method for synthesizing levofolinate based on an enzyme-catalyzed tandem reaction.

[0011] The technical solution of the present application to solve the above problems is as follows:

[0012] A method for synthesizing levofolinate based on an enzyme-catalyzed tandem reaction, which uses folic acid as a substrate in a reaction system containing dihydrofolate reductase, tetrahydrofolate formyltransferase, and an NAD(P)-dependent coenzyme cycle system, and obtains the product levofolinate through enzyme-catalyzed reaction.

[0013] The dihydrofolate reductase has an amino acid sequence as shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, or SEQ ID NO. 8, or has at least 80% identity with the sequence shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, or SEQ ID NO. 8.

[0014] As a preferred embodiment of the above technical solution, the gene encoding dihydrofolate reductase is derived from one of the following: LbuDHFR of Lactobacillus bulgaricus, BhaDHFR of Bacillus halodurans, BloDHFR of Bifidobacterium longum, and BinDHFR of Bifidobacterium infantis.

[0015] As a preferred embodiment of the above technical solution, the tetrahydrofolate formyltransferase has an amino acid sequence as shown in SEQ ID NO. 10 and SEQ ID NO. 12, or has at least 80% identity with the sequence shown in SEQ ID NO. 10 and SEQ ID NO. 12.

[0016] As a preferred embodiment of the above technical solution, the encoding gene of the tetrahydrofolate formyltransferase is shown in SEQ ID NO. 9 and SEQ ID NO. 11.

[0017] As a preferred embodiment of the above technical solution, the gene encoding the tetrahydrofolate formyltransferase is derived from either TacFTCD of *Thermogymnomonas acidicola* or TthFTCD of *Thermoanaerobacterium thermosaccharolyticum*.

[0018] This invention relates to the synthesis of levofolate based on an enzymatic tandem reaction. In the method of this invention, the substrate folic acid in the system first undergoes an asymmetric hydrogenation reaction under the synergistic catalysis of dihydrofolate reductase (DHFR) and NAD(P)-dependent coenzyme cycle to obtain L-tetrahydrofolate (6S-THF), which is then catalyzed by tetrahydrofolate formyltransferase (FTCD) to obtain optically pure levofolate.

[0019] The method for synthesizing levofolate in this invention is based on an enzymatic tandem reaction and is a one-pot process, eliminating the need for intermediate product separation. Specifically, the intermediate 6S-THF produced by the dihydrofolate reductase-catalyzed reaction can be immediately converted to levofolate by tetrahydrofolate-formyltransferase. The intermediate product is produced and used immediately without separation, thus solving the problem of (intermediate) product inhibition. In a specific example of this invention, using 2 g / L folic acid as a substrate, the catalytic reaction was carried out for 10 h, and the substrate conversion rate was greater than 99%.

[0020] As a preferred embodiment of the above technical solution, the temperature of the enzyme-catalyzed reaction is 25~40℃ and the pH is 7.0~9.0.

[0021] As a preferred embodiment of the above technical solution, N-formyl-L-glutamic acid is added to the reaction system as a formyl donor substrate.

[0022] This invention relates to a method for synthesizing levofolate based on an enzymatic tandem reaction, which requires NAD+. + or NADP + Coenzymes are NAD(P)-dependent dehydrogenases and their substrates forming a coenzyme cycle. Preferred combinations of dehydrogenases and substrates within this coenzyme cycle include alcohol dehydrogenase / isopropanol, glucose dehydrogenase / glucose, and formate dehydrogenase / sodium formate.

[0023] In summary, the present invention has the following beneficial effects:

[0024] 1. The method of the present invention uses a whole-enzyme method to catalyze the conversion of folic acid into levofolinic acid, which is mild and avoids the safety hazards of the chemical method for synthesizing levofolinic acid commonly used in the prior art.

[0025] 2. The method of the present invention uses a one-pot process, which avoids the intermediate product separation step in the chemical synthesis process. In this method, the intermediate generated is converted into the product levofloxacin in a timely manner, which can be used immediately after production, and solves the problem of product inhibition in the chemical method.

[0026] 3. The method of the present invention uses an asymmetric synthesis process of biological enzymes, which avoids the diastereomer separation step in the chemical synthesis process, simplifies the steps, effectively reduces costs, and improves conversion yield.

[0027] 4. Compared with existing chemical methods for synthesizing leucovorin, the method of the present invention is simpler and has a higher conversion yield. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the structure of the recombinant plasmid pET28a-LbuDHFR;

[0029] Figure 2 This is an SDS-PAGE analysis diagram of dihydrofolate reductase expression in Example 2 of the present invention;

[0030] Figure 3 This is the HPLC chromatogram of the DHFR-catalyzed conversion of folic acid to L-tetrahydrofolate in Example 2 of the present invention;

[0031] Figure 4 This is a schematic diagram of the structure of the recombinant plasmid pET28a-TthFTCD;

[0032] Figure 5 This is an SDS-PAGE analysis diagram of tetrahydrofolate formyltransferase expression in Example 4 of the present invention;

[0033] Figure 6 This is the HPLC chromatogram of L-tetrahydrofolate formylation catalyzed by FTCD in Example 4 of the present invention;

[0034] Figure 7 This is the HPLC chromatogram of the reaction for preparing levofolinic acid from folic acid in Example 7 of the present invention after 2 hours.

[0035] Figure 8 This is a process route diagram for a method of synthesizing levofolinic acid based on an enzymatic tandem reaction according to the present invention. Detailed Implementation

[0036] The present invention will be further described below with reference to embodiments.

[0037] In the examples, the LB medium used consisted of 5 g / L yeast extract, 10 g / L peptone, and 10 g / L NaCl, with deionized water as the solvent and a pH of 6.8–7.0. The final concentration of the fermentation medium was: 12 g / L yeast extract, 15 g / L peptone, 10 g / L glycerol, 8.9 g / L Na₂HPO₄·12H₂O, 3.4 g / L KH₂PO₄, 2.67 g / L NH₄Cl, 0.71 g / L Na₂SO₄, and 0.3 g / L MgSO₄·7H₂O, with deionized water as the solvent and a pH of 6.8–7.0.

[0038] Example 1: Screening for highly active dihydrofolate reductase

[0039] Inducible expression of dihydrofolate reductase.

[0040] Based on our previously constructed dihydrofolate reductase resource library (React. Chem. Eng., 2024, 9, 3110), cryopreserved glycerol tube seed cultures from various engineered strains were extracted, activated, and then fermented and induced to express. The seed culture was activated by streaking on LB agar plates containing 50 mg / L kanamycin and incubated overnight at 37°C. Freshly cultured single colonies were picked and inoculated into LB liquid medium containing 50 mg / L kanamycin and incubated at 37°C and 200 rpm for 12 h. The freshly activated seed culture was then inoculated at a 1% (v / v) inoculation rate into LB liquid medium containing 50 mg / L kanamycin and incubated at 37°C and 200 rpm for 4 h to obtain freshly cultured seed culture. The seed culture was inoculated into a fermentation medium containing 50 mg / L kanamycin at a seeding rate of 2% (v / v) and cultured at 37°C and 200 rpm for 3 h on a shaker. IPTG was added to a final concentration of 0.5 mmol / L and cultured at 24°C and 200 rpm for 8 h to obtain the fermentation broth of dihydrofolate reductase.

[0041] Comparison of the catalytic activity of dihydrofolate reductase in catalytic reaction systems containing folinic acid.

[0042] The dihydrofolate reductase fermentation broth was centrifuged at 8000×g for 10 min at 4℃, and the supernatant was removed. The cells were resuspended in 400 mM HEPES (pH 7.5) buffer, and the cells were homogenized using a high-pressure cell homogenizer to obtain crude enzyme solution. It should be used for catalytic reaction as soon as possible to avoid long-term storage.

[0043] The crude enzyme was used for SDS-PAGE electrophoresis analysis to determine the induced expression of dihydrofolate reductase. The results are as follows: Figure 2 As shown.

[0044] The catalytic activity of dihydrofolate reductase for folic acid in a folinic acid-containing catalytic reaction system was analyzed. To the crude enzyme solution of dihydrofolate reductase, 25 mmol / L L-ascorbic acid, 6 mmol / L dithiothreitol (DTT), 1.0 g / L folic acid, 1.0 g / L folinic acid, and 10 mmol / L NADPH were added sequentially. The reaction solution was placed in a 20 mL round-bottom flask, magnetically stirred, and reacted at 37 °C for 1 h. Under magnetic stirring, 500 μL of the reaction solution was sampled and centrifuged in 500 μL of 5% ammonia water at 10000 × g for 5 min. The supernatant was used for liquid chromatography analysis.

[0045] Liquid chromatography analysis method: Column: C 18 Column; Detection wavelength: 290 nm; Mobile phase: phosphate buffer (pH 6.3); Flow rate: 1.0 mL / min; Column temperature: 40℃; Injection volume: 10 μL. Dihydrofolate reductase catalyzes the asymmetric hydrogenation reaction of folic acid to produce L-tetrahydrofolate. The HPLC chromatogram of the catalytic process is shown below. Figure 3 As shown, the retention time of the substrate folic acid was 8.335 min; the retention time of the product L-tetrahydrofolate was 4.372 min.

[0046] The catalytic activities of dihydrofolate reductase are compared in Table 1.

[0047] Table 1. Comparison of catalytic activities of dihydrofolate reductase in catalytic reaction systems containing folinic acid.

[0048] SEQ ID NO: Name of enzyme Conversion of 1.0 g / L folic acid substrate in a catalytic reaction system containing 1.0 g / L folinic acid, % 1 AmaDHFR2855 ND (no product detected) 2 AmaDHFR2417 ND 3 AmaDHFR0532 ND 4 AmaDHFR5858 ND 5 BsuDHFR168 ND 6 XcaDHFR ND 7 AjuDHFR ND 8 SmuDHFR ND 9 SceDHFR ND 10 KlaDHFR ND 11 AorDHFR ND 12 LbrDHFR0785 ND 13 LbrDHFR1497 ND 14 SthDHFR ND 15 BbiDHFR 3% 16 LplDHFR ND 17 EcoDHFR ND 18 EcoDHFR ANLYF ND 19 hDHFR-35K64F ND 20 PfDHFR ND 21 BhaDHFR 5.91% 22 TransDHFR ND 23 OihDHFR ND 24 LasDHFR ND 25 KcoDHFR ND 26 PpaDHFR ND 27 BinDHFR 3.51% 28 BloDHFR 3.43% 29 BbrDHFR 1.85% 30 LheDHFR ND 31 LrhDHFR 2.73% 32 BanDHFR ND 33 LbuDHFR 16.66% 34 LacDHFR ND 35 CamDHFR ND 36 CsaDHFR ND 37 BcaDHFR ND

[0049] Among them, the dihydrofolate reductases with higher enzyme activity are LbuDHFR from Lactobacillus bulgaricus, BhaDHFR from Bacillus halodurans, BloDHFR from Bifidobacterium longum, and BinDHFR from Bifidobacterium infantis, with LbuDHFR exhibiting the highest catalytic activity.

[0050] Example 2: Construction of recombinant Escherichia coli engineered strain of tetrahydrofolate formyltransferase FTCD

[0051] Screening and synthesis of tetrahydrofolate formyltransferase gene.

[0052] By utilizing the KEGG and NCBI databases and combining sequence homology comparative analysis, six tetrahydrofolate formyltransferases were identified, including SpyFTCD from *Streptococcus pyogenes*, TacFTCD from *Thermogymnomonas acidicola*, TthFTCD from *Thermoanaerobacterium thermosaccharolyticum*, AmeFTCD from *Alkaliphilus metalliredigens*, FacFTCD from *Ferroplasma acididiphilum*, and SacFTCD from *Sulphobic Bacillus*. These FTCD enzymes were recombinantly expressed, and their tetrahydrofolate transformylation activities under conditions containing a certain concentration of folic acid were compared, specifically the catalytic activity of converting 6S-tetrahydrofolate (compound III) to L-5-formyltetrahydrofolate (compound I).

[0053] Recombinant expression strains were constructed targeting the six tetrahydrofolate-formyltransferases. First, their corresponding amino acid sequences were submitted to a gene synthesis company (Suzhou Genewiz Biotechnology Co., Ltd.), where the gene sequences of SpyFTCD, TacFTCD, TthFTCD, AmeFTCD, FacFTCD, and SacFTCD were artificially synthesized. These genes were then cloned into the NdeI and BamHI regions of the expression vector pET28a, respectively, to obtain recombinant plasmids pET28a-AmeFTCD, pET28a-FacFTCD, pET28a-SacFTCD, pET28a-SpyFTCD, pET28a-TacFTCD, and pET28a-TthFTCD. The map of the recombinant plasmid pET28a-TthFTCD is shown below. Figure 4 As shown in the diagram. These recombinant plasmids were transformed into *E. coli* BL21(DE3) strain to construct the corresponding recombinant expression strains. The structural diagram of the recombinant plasmid pET28a-LbuDHFR is shown below. Figure 1 As shown.

[0054] Example 3: Induced expression and enzyme activity analysis of tetrahydrofolate formyltransferase

[0055] Freshly streaked single colonies of the recombinant *E. coli* strains expressing tetrahydrofolate transferases from different sources, constructed in Example 2, were inoculated into LB medium containing 50 mg / L kanamycin and cultured overnight at 37°C and 200 rpm. The activated seed culture was then inoculated at a rate of 1% (v / v) into LB liquid medium containing 50 mg / L kanamycin and cultured at 37°C and 200 rpm for 4 h to obtain freshly cultured seed culture.

[0056] Freshly cultured seed culture was inoculated into E. coli fermentation medium containing the corresponding resistance (50 mg / L kanamycin) at a volume concentration of 2%. The culture was incubated at 37°C for 3 h. When the OD600 of the cells reached 0.6-0.8, IPTG was added to a final concentration of 0.25 mmol / L. The fermentation temperature was controlled at 24°C, and fermentation was continued for 10 h to obtain the fermentation broth of tetrahydrofolate forsylate transferase.

[0057] The centrifuged fermentation broth was resuspended in pH 7.5, 400 mM HEPES buffer, and the cells were disrupted using a high-pressure cell homogenizer to obtain crude enzyme solution. This solution should be used for catalytic reactions as soon as possible to avoid long-term storage.

[0058] The crude enzyme was used for SDS-PAGE electrophoresis analysis to determine the induced expression of tetrahydrofolate formyltransferase. The results are as follows: Figure 5 As shown.

[0059] Comparative analysis of the catalytic activity of tetrahydrofolate formyltransferase. To the crude tetrahydrofolate formyltransferase solution, N-formyl-L-glutamic acid (5.7 mM), 6 mM DTT, 1.0 g / L L-tetrahydrofolate, and 1.0 g / L folic acid were added sequentially. The reaction solution was placed in a 20 mL round-bottom flask, magnetically stirred, and reacted at 37 °C for 1 h. Under magnetic stirring, 500 μL of the reaction solution was added to 500 μL of 5% ammonia water, centrifuged at 10000 × g for 5 min, and the supernatant was used for liquid chromatography analysis. Liquid chromatography analysis method: Column: C18 column; Detection wavelength: 290 nm; Mobile phase: phosphate buffer (pH 6.3); Flow rate: 1.0 mL / min; Column temperature: 40 °C; Injection volume: 10 μL. The HPLC chromatogram of the catalytic process is shown below. Figure 6 As shown, the retention time of the product L-5-formyltetrahydrofolate was 5.245 min.

[0060] The catalytic activities of various tetrahydrofolate transferases are compared in Table 2. Table 2: Comparison of catalytic activities of recombinant tetrahydrofolate transferase strains.

[0061] SEQ ID NO: Number NBCI Accession Number Conversion of 1 g / L tetrahydrofolate substrate under conditions containing 1 g / L folic acid, % 1 AmeFTCD WP_012065545.1 ND 2 FacFTCD WP_309209531.1 ND 3 SacFTCD MCY0864488.1 38.20% 4 SpyFTCD MEN4364178.1 50.58% 5 TacFTCD WP_188679644.1 81.09% 6 TthFTCD WP_015312349.1 83.57%

[0062] As can be seen from Table 2, their catalytic activity ranks as follows: TthFTCD > TacFTCD > SpyFTCD > SacFTCD.

[0063] The nucleotide sequences of the TacFTCD and TthFTCD genes are shown in SEQ ID NO.9 and SEQ ID NO.11, respectively. The amino acid sequences encoded by TacFTCD and TthFTCD are shown in SEQ ID NO.10 and SEQ ID NO.12, respectively.

[0064] Example 4: Establishment of a three-enzyme coupling reaction of dihydrofolate reductase DHFR, tetrahydrofolate methyltransferase FTCD, and NADP-dependent coenzyme cycle system.

[0065] Based on the catalytic mechanism of dihydrofolate reductase, the coenzyme NAD(P)H is required. To improve the catalytic efficiency of the one-pot enzymatic preparation of leucovorin, NAD(P)H is employed. + The / NAD(P)H coenzyme cycle system was used in this catalytic reaction system. The alcohol dehydrogenase lbADH from *Lactobacillus brevis* was used as the coenzyme cycle system in subsequent examples.

[0066] The crude enzyme solution of alcohol dehydrogenase lbADH was prepared as follows. The recombinant *E. coli* IEF-lbADH expressing lbADH enzyme was constructed in our previous study (CN112143764A). Cryopreserved glycerol seed tubes were taken and activated by streaking on LB agar plates containing 50 mg / L kanamycin. Freshly cultured single colonies were picked and inoculated into LB medium containing 50 mg / L kanamycin, and incubated overnight at 37°C and 200 rpm. The activated seed solution was then inoculated at a 1% (v / v) inoculation rate into LB liquid medium containing 50 mg / L kanamycin, and incubated at 37°C and 200 rpm for 4 h to obtain the freshly cultured seed solution.

[0067] Freshly cultured seed culture was inoculated into Escherichia coli fermentation medium containing 50 mg / L kanamycin at a volume concentration of 2%, and cultured at 37°C for 3 h. When the OD600 of the cells reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mmol / L, and the fermentation temperature was controlled at 24°C. Fermentation was continued for 10 h to obtain 1bADH fermentation broth.

[0068] Leucovorin was synthesized using a one-pot, three-enzyme catalytic reaction with TacFTCD. The TacFTCD fermentation broth from Example 3, the LbuDHFR fermentation broth from Example 1, and the aforementioned lbADH fermentation broth were mixed in a total OD600 ratio of TacFTCD:LbuDHFR:lbADH = 3:2:1 to obtain a three-enzyme mixed fermentation broth. The mixed fermentation broth was centrifuged (8000×g, 10 min, 4℃), the supernatant was removed, and the cells were resuspended in pH 7.5, 400 mM HEPES buffer until the cell density OD600 reached 10. Cells were then homogenized using a high-pressure cell homogenizer to obtain a crude enzyme solution containing the three enzymes. This solution should be used for the catalytic reaction as soon as possible to avoid prolonged storage. The following three-enzyme reaction system was prepared with a final concentration of 0.1 mM NADP. + The reaction mixture consisted of 3% (v / v) isopropanol, 6 mM DTT, 5.7 mM N-formyl-L-glutamic acid, and 1.0 g / L folic acid (compound II), with the volume made up to 200 ml with deionized water. The pH was then adjusted to 7.5. The reaction system was subjected to magnetic stirring in a 37°C water bath for 5 h. The reaction solution was analyzed by HPLC, and the results showed that some of the substrates, compound II (folic acid) and compound III (L-tetrahydrofolic acid), remained unconverted, with a final conversion rate of 89%.

[0069] Leucovorin was synthesized using a one-pot, three-enzyme catalytic reaction with TthFTCD. The TthFTCD fermentation broth from Example 3, the LbuDHFR fermentation broth from Example 1, and the aforementioned lbADH fermentation broth were mixed in a total OD600 ratio of TthFTCD:LbuDHFR:lbADH = 3:2:1 to obtain a three-enzyme mixed fermentation broth. The mixed fermentation broth was centrifuged (8000×g, 10 min, 4℃), the supernatant was removed, and the cells were resuspended in pH 7.5, 400 mM HEPES buffer until the cell density OD600 reached 10. After cell homogenization using a high-pressure cell homogenizer, a crude enzyme solution was obtained, which should be used for the catalytic reaction as soon as possible to avoid prolonged storage. The following three-enzyme reaction system was prepared with a final concentration of 0.1 mM NADP. + The reaction mixture consisted of 3% (v / v) isopropanol, 6 mM DTT, 5.7 mM N-formyl-L-glutamic acid, and 1.0 g / L folic acid (compound II), with the volume made up to 200 ml with deionized water. The pH was then adjusted to 7.5. The reaction system was subjected to magnetic stirring in a 37°C water bath for 5 h. The reaction solution was analyzed by HPLC, and the results showed that some of the substrates, compound II (folic acid) and compound III (L-tetrahydrofolic acid), remained unconverted, with a final conversion rate of 95%.

[0070] Example 5: Preparation of L-5-formyltetrahydrofolate by mixing LbuDHFR, lbADH and TthFTCD fermentation broth

[0071] A method for synthesizing levofolate based on an enzymatic tandem reaction, the process route is as follows: Figure 8 As shown.

[0072] Catalytic conversion of 1.0 g / L folic acid substrate.

[0073] Centrifuge the LbuDHFR fermentation broth prepared by the method in Example 1 and collect 10.0 g of wet cell culture; centrifuge the lbADH fermentation broth prepared by the method in Example 4 and collect 5.0 g of wet cell culture; centrifuge the TthFTCD fermentation broth prepared by the method in Example 3 and collect 15.0 g of wet cell culture. Resuspend the three types of wet cell culture in 150 mL of pH 7.5, 400 mM HEPES buffer, homogenize the cells using a high-pressure homogenizer, and add 0.1 mM NADP to a final concentration. +3% (v / v) isopropanol, 6 mM DTT, 10 mM N-formyl-L-glutamic acid, and 1.0 g / L folic acid (compound II) were added to a final volume of 200 ml with deionized water, and the pH was adjusted to 7.5. The reaction system was subjected to magnetic stirring in a 37°C water bath for 12 h. The reaction solution was analyzed by HPLC, and the results showed that some of the substrates, compound II (folic acid) and compound III (L-tetrahydrofolic acid), remained unconverted, with a final conversion rate of 99%.

[0074] Catalytic conversion of folic acid substrate at a concentration of 2.0 g / L.

[0075] Centrifuge the LbuDHFR fermentation broth prepared by the method in Example 1 and collect 20.0 g of wet cell culture; centrifuge the lbADH fermentation broth prepared by the method in Example 4 and collect 10.0 g of wet cell culture; centrifuge the TthFTCD fermentation broth prepared by the method in Example 3 and collect 30.0 g of wet cell culture. Resuspend the three types of wet cell culture in 150 mL of pH 7.5, 400 mM HEPES buffer, homogenize the cells using a high-pressure homogenizer, and add 0.1 mM NADP to a final concentration. + 3% (v / v) isopropanol, 6 mM MDT, 20 mM N-formyl-L-glutamic acid, and 2.0 g / L folic acid (compound II) were added to a final volume of 200 ml with deionized water, and the pH was adjusted to 7.5. The reaction system was subjected to magnetic stirring in a 37°C water bath for 12 h. The reaction solution was analyzed by HPLC, and the results showed that some of the substrates, compound II (folic acid) and compound III (L-tetrahydrofolic acid), remained unconverted, with a final conversion rate of 99%.

[0076] Catalytic conversion of folic acid substrate at 3.0 g / L.

[0077] Centrifuge the LbuDHFR fermentation broth prepared by the method in Example 1 and collect 30.0 g of wet cell culture; centrifuge the lbADH fermentation broth prepared by the method in Example 4 and collect 15.0 g of wet cell culture; centrifuge the TthFTCD fermentation broth prepared by the method in Example 3 and collect 45.0 g of wet cell culture. Resuspend the three types of wet cell culture in 150 mL of pH 7.5, 400 mM HEPES buffer, homogenize the cells using a high-pressure homogenizer, and add 0.1 mM NADP to a final concentration. +The reaction mixture consisted of 3% (v / v) isopropanol, 6 mM MDT, 30 mM N-formyl-L-glutamic acid, and 3.0 g / L folic acid (compound II), with the volume made up to 200 ml with deionized water. The pH was then adjusted to 7.5. The reaction system was subjected to magnetic stirring in a 37°C water bath for 12 h. The reaction solution was analyzed by HPLC, and the results showed that some of the substrates, compound II (folic acid) and compound III (L-tetrahydrofolic acid), remained unconverted, with a final conversion rate of 95%.

[0078] Catalytic conversion of 5.0 g / L folic acid substrate.

[0079] Centrifuge the LbuDHFR fermentation broth prepared by the method in Example 1 and collect 40.0 g of wet cell culture; centrifuge the lbADH fermentation broth prepared by the method in Example 4 and collect 20.0 g of wet cell culture; centrifuge the TthFTCD fermentation broth prepared by the method in Example 3 and collect 60.0 g of wet cell culture. Resuspend the three types of wet cell culture in 150 mL of pH 7.5, 400 mM HEPES buffer, homogenize the cells using a high-pressure homogenizer, and add 0.1 mM NADP to a final concentration. + 3% (v / v) isopropanol, 6 mM MDT, 50 mM N-formyl-L-glutamic acid, and 5.0 g / L folic acid (compound II) were added to a final volume of 200 ml with deionized water, and the pH was adjusted to 7.5. The reaction system was subjected to magnetic stirring in a 37°C water bath for 12 h. The reaction solution was analyzed by HPLC, and the results showed that some of the substrates, compound II (folic acid) and compound III (L-tetrahydrofolic acid), remained unconverted, with a final conversion rate of 82%.

[0080] The sampling and HPLC analysis method is as follows: Under magnetic stirring, 500 μL of the reaction solution was added to 500 μL of 5% ammonia water, centrifuged at 10000×g for 5 min, and the supernatant was used for HPLC analysis. HPLC analysis method: Column: C18 column; Detection wavelength: 254 nm and 290 nm; Mobile phase: phosphate buffer (pH 6.3); Flow rate: 1.0 mL / min; Column temperature: 40℃; Injection volume: 10 μL. The HPLC chromatogram of the catalytic process is shown below. Figure 7 As shown, the retention time of the product L-5-formyltetrahydrofolate was 5.173 min.

[0081] Analysis and evaluation

[0082] Analysis of the three-enzyme catalytic reaction solution in Example 5 shows that the three-enzyme coupling reaction of dihydrofolate reductase LbuDHFR, tetrahydrofolate formyltransferase TthFTCD, and alcohol dehydrogenase, after 10-12 hours of catalysis, can produce a product, leucovorin, at a concentration greater than 2 g / L, with a conversion rate of folic acid greater than 99%. The one-pot method of this invention for preparing L-5-formyltetrahydrofolate has a high yield and significant application value and market potential.

[0083] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for synthesizing levofolinate based on an enzymatic cascade reaction, characterized by: In a reaction system containing dihydrofolate reductase, tetrahydrofolate formyltransferase and NAD(P)-dependent coenzyme cycle system, the product levofolinate is obtained by enzyme catalytic reaction with folate as substrate; The dihydrofolate reductase has an amino acid sequence as shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 8, or has at least 80% identity with the sequence as shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO.

8.

2. The method for synthesizing levo-folic acid based on enzymatic tandem reaction according to claim 1, characterized in that: The coding gene of the dihydrofolate reductase is derived from one of LbuDHFR of Lactobacillus bulgaricus, BhaDHFR of Bacillus halodurans, BloDHFR of Bifidobacterium longum and BinDHFR of Bifidobacterium infantis.

3. The method for synthesizing levo-folic acid based on enzymatic tandem reaction according to claim 1 or 2, characterized in that: The tetrahydrofolate formyltransferase has an amino acid sequence as shown in SEQ ID NO. 10 or SEQ ID NO. 12, or has at least 80% identity with the sequence as shown in SEQ ID NO. 10 or SEQ ID NO.

12.

4. The method for synthesizing levo-folic acid based on enzymatic tandem reaction according to claim 3, characterized in that: The coding gene of the tetrahydrofolate formyltransferase is as shown in SEQ ID NO. 9 or SEQ ID NO.

11.

5. The method for synthesizing levo-folic acid based on enzymatic tandem reaction according to claim 4, characterized in that: The coding gene of the tetrahydrofolate formyltransferase is derived from one of TacFTCD of Thermogymnomonas acidicola and TthFTCD of Thermoanaerobacterium thermosaccharolyticum.

6. The method of claim 1, wherein the method is based on an enzymatic cascade reaction. The temperature of the enzyme catalytic reaction is 25-40℃, and the pH is 7.0-9.0.

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