A tetrahydrofolate methyltransferase mutant, encoding gene and application thereof

Abstract

The application belongs to the field of genetic engineering and enzyme engineering, and particularly relates to a tetrahydrofolate methyltransferase mutant, a coding gene and application thereof. The application provides a tetrahydrofolate methyltransferase RcoDmdA mutant, and the amino acid sequence of the mutant is shown in one of SEQ ID NO. 3-9. The RcoDmdA mutant provided by the application has higher catalytic activity, the substrate THF feeding amount is significantly increased to 15-20 g / L, the yield of the product L-5-MTHF is increased to 15-17 g / L, the substrate conversion rate is high, and the tetrahydrofolate methyltransferase mutant and the coding gene provided by the application have important industrial application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering and enzyme engineering, and particularly relates to a tetrahydrofolate methyltransferase mutant, a coding gene and application thereof. BACKGROUND

[0002] L-5-methyltetrahydrofolate (L-5-MTHF) is the natural active form of folic acid (FA) and the main folate component in the blood circulation system, which can be directly absorbed and utilized. It has been recommended as a substitute for synthetic folic acid. The advantage of L-5-MTHF as a nutritional supplement is that it has higher bioavailability and can be directly absorbed and utilized without metabolic conversion, thereby bypassing the DHFR and MTHFR gene polymorphism and addressing the problem of insufficient absorption and utilization of synthetic folic acid. Active folic acid L-5-MTHF has the properties of natural folic acid and does not accumulate in the blood as unmetabolized synthetic folic acid.

[0003] Due to the high bioavailability of L-5-MTHF, its stable structure L-5-methyltetrahydrofolate calcium was approved by the US FDA in 2001 and applied as a new raw material in nutritional supplements. Subsequently, it entered the international market as a safer folic acid food additive. Currently, L-5-MTHF, as an innovative folic acid drug, has gradually replaced chemically synthesized FA and is widely used in the fields of medicine, food, and animal husbandry. Existing L-5-MTHF products are prepared by chemical methods, i.e., using synthetic folic acid as the starting material, preparing tetrahydrofolate by chemical hydrogenation, obtaining (6S, αS) tetrahydrofolate through crystallization separation, and further methylating and reducing to obtain (6S, αS)-5-MTHF (L-5-MTHF) with physiological activity. The reaction process is complex, the preparation cost is high, and it contains impurity components of (6R, αS)-5-MTHF which do not have physiological activity.

[0004] With the rapid development of biotechnology, green and environmentally friendly production methods with high efficiency and low cost have gradually attracted people's attention. Among them, the green synthesis technology of L-5-MTHF by biological enzyme catalysis has also been continuously explored and improved, including biological fermentation method (Microb. Biotechnol. 2022, 15:2758-2772, J. Agric. Food. Chem. 2022, 70:5849-5859) and enzyme catalysis method (CN116574768A). Biological enzyme method has the characteristics of high catalytic efficiency, high selectivity, mild reaction conditions, low energy consumption, and no toxicity, which meets the green development direction. Therefore, the biological enzyme method based on high-activity biological enzymes has important application value.

[0005] The present disclosure provides a tetrahydrofolate methyltransferase mutant derived from Ruegeria conchae, which can catalyze efficient methylation of tetrahydrofolate (THF) to generate L-5-MTHF, and has the characteristics of high substrate amount and fast catalytic reaction rate. SUMMARY

[0006] In order to overcome the problems of low catalytic efficiency, low yield and low conversion rate in the prior art, the present application provides a tetrahydrofolate methyltransferase mutant, a coding gene and applications thereof.

[0007] In order to achieve the above-mentioned purpose, the present application realizes the following technical scheme:

[0008] In a first aspect, the present application provides a tetrahydrofolate methyltransferase RcoDmdA mutant, wherein the amino acid sequence of the mutant is shown in one of SEQ ID NO. 3-9.

[0009] The present application mutates and modifies a tetrahydrofolate methyltransferase RcoDmdA derived from Ruegeria conchae to improve its catalytic activity, and the amino acid sequence of RcoDmdA is shown in SEQ ID NO. 2.

[0010] The present application mutates and modifies the amino acid residue sites of the methyl acceptor substrate THF binding domain of RcoDmdA, including the V121, P150, S196, F245, M249 and F263 sites, and the amino acid sites on the methyl donor substrate dimethyl mercaptopropionic acid DMSP binding domain, including the Y29, C60 and G249 sites.

[0011] The present application uses computer-aided software to perform homology modeling on the wild-type RcoDmdA, and compares and analyzes the known homologous crystal structure, i.e., the tetrahydrofolate methyltransferase PubDmdA (PDB ID: 3TFI and 3TFJ) derived from Pelagibacter ubique, identifies and screens the key amino acid residue sites, and the results are shown in Figure 2 The mutant enzyme is obtained by site-directed substitution mutation, and an excellent mutant is screened.

[0012] The present application mutates and modifies 13 amino acid residues in the THF binding pocket of RcoDmdA, including Y29, Y93, P107, V121, A122, P150, F175, F176, S193, G194, L242, L243 and F263. The present application constructs a small and precise combination mutation library, i.e., a three-codon saturation mutation technology, screens and obtains an excellent mutant.

[0013] This invention divides the 13 selected amino acid residues into four groups: group A (Y29, Y93, P107), group B (V121, A122, F175, F176), group C (P150, S193, G194), and group D (L242, L243, F263), and follows the order described above. Figure 4 The proposed scheme constructs four small, high-quality libraries. The three chosen codons are isoleucine, serine, and tyrosine, used for three-codon saturation mutations at each site. Superior mutants are obtained through screening of each mutant library.

[0014] The amino acid sequences of the superior mutants described in this invention are shown in SEQ ID NO. 3 to 9, namely RcoDmdA-F263Y, RcoDmdA-V121I, RcoDmdA-A122S, RcoDmdA-V121S, RcoDmdA-P107I, RcoDmdA-F263I, and RcoDmdA-F245Y, respectively.

[0015] Secondly, the present invention provides a gene for a tetrahydrofolate methyltransferase RcoDmdA mutant.

[0016] Preferably, the nucleotide sequence of the encoding gene is shown in one of SEQ ID NO. 10 to 16.

[0017] The coding genes of the superior mutants F263Y, V121I, A122S, V121S, P107I, F263I, and F245Y described in this invention can be synthesized by codon optimization based on their amino acid sequences. Preferably, the nucleotide sequences of the coding genes are shown in SEQ ID NO. 10-16.

[0018] Thirdly, the present invention also provides a recombinant vector for the encoded gene.

[0019] Fourthly, the present invention also relates to genetically engineered bacteria that encode the aforementioned gene.

[0020] Fifthly, the present invention also provides the application of the tetrahydrofolate methyltransferase RcoDmdA mutant in the enzyme-catalyzed preparation of L-5-methyltetrahydrofolate from tetrahydrofolate (THF).

[0021] Preferably, the application constructs a genetically engineered bacterium containing the mutant encoding gene, and uses wet bacterial cells obtained by fermentation culture of the genetically engineered bacterium or enzyme-containing cells obtained by cell disruption as a catalyst to methylate THF to obtain L-5-MTHF.

[0022] The present invention can clone the genes of the RcoDmdA mutants F263Y, V121I, A122S, V121S, P107I, F263I and F245Y into expression plasmids, transform them into host cells, and obtain enzyme catalysts through induced fermentation, which are used to catalyze the conversion of THF to L-5-MTHF.

[0023] As a preferred embodiment, the pET28a plasmid and Escherichia coli BL21 host cells were used to construct recombinant E. coli BL21.

[0024] The expression plasmid and host cell described in this invention are preferably pET28a plasmid and Escherichia coli BL21(DE3) host cells, i.e., constructing recombinant bacteria E. coli BL21(DE3) (pET28a-RcoDmdA-F263Y, pET28a-RcoDmdA-V121I, pET28a-RcoDmdA-A122S, pET28a-RcoDmdA-V121S, pET28a-RcoDmdA-P107I, pET28a-RcoDmdA-F263I, pET28a-RcoDmdA-F245Y).

[0025] Preferably, the catalytic activity is carried out under conditions of adding the methyl donor dimethyl sulfonium chloride.

[0026] The present invention relates to a method for using wet bacterial cells or cell lysate supernatant obtained by cell disruption to catalyze the conversion of THF to L-5-MTHF, characterized by the addition of cell density OD. 600 The concentration of the substrate is 20 to 80 (preferably 40), the amount of the substrate THF is 1 g / L to 20 g / L (preferably 10-15 g / L), and the amount of the methyl donor substrate dimethyl sulfonium chloride is 10 mM to 300 mM (preferably 150 mM).

[0027] Preferably, the catalytic reaction is carried out at a pH of 6.0-9.0 and a reaction temperature of 25℃-50℃ to obtain the product L-5-MTHF.

[0028] The present invention catalyzes a reaction at a pH of 6.0-9.0 (preferably 7.5) and a reaction temperature of 25℃-50℃ (preferably 37℃) to obtain the product L-5-MTHF with a conversion rate of 85%-99%.

[0029] The beneficial effects of this invention are mainly reflected in:

[0030] (1) The RcoDmdA mutant provided by this invention has higher catalytic activity;

[0031] (2) The amount of substrate THF was significantly increased to 15-20 g / L, and the yield of product L-5-MTHF was increased to 15-17 g / L, resulting in high substrate conversion.

[0032] (3) The tetrahydrofolate methyltransferase mutant and its encoding gene provided by the present invention have important industrial application value. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the structure of wild-type tetrahydrofolate methyltransferase RcoDmdA.

[0034] Figure 2 This is a schematic diagram of the amino acid residue sites around the binding pocket of wild-type tetrahydrofolate methyltransferase RcoDmdA with THF and methyl donor DMSP.

[0035] Figure 3 Wild-type tetrahydrofolate methyltransferase RcoDmdA and THF surrounding A schematic diagram of the 13 unsaturated amino acid sites within the range.

[0036] Figure 4 A schematic diagram illustrating the grouping and construction of small, refined libraries for three-codon saturation mutations.

[0037] Figure 5 This is a schematic diagram showing the initial screening results of the A group mutant library in the first-generation three-codon saturated mutant library.

[0038] Figure 6 This is a schematic diagram showing the initial screening results of the B group mutant library in the first-generation three-codon saturated mutant library.

[0039] Figure 7 This is a schematic diagram showing the initial screening results of the C group mutant library in the first-generation three-codon saturated mutant library.

[0040] Figure 8 This is a schematic diagram showing the initial screening results of the D group mutant library in the first-generation three-codon saturated mutant library.

[0041] Figure 9 Comparison of product synthesis curves for the preparation of L-5-MTHF from THF catalyzed by single-point mutants.

[0042] Figure 10 HPLC chromatograms of THF and L-5-MTHF standard samples. Detailed Implementation

[0043] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0044] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0045] LB medium: yeast extract 5.0 g / L, peptone 10.0 g / L, NaCl 10.0 g / L, solvent is distilled water.

[0046] Fermentation medium: yeast extract 12.0 g / L, peptone 15.0 g / L, Na2HPO4·12H2O 8.9 g / L, KH2PO4 3.4 g / L, NH4Cl 2.67 g / L, Na2SO4 0.71 g / L, MgSO4·7H2O 0.49 g / L, kanamycin 50 μg / L, pH 7.0, solvent: distilled water.

[0047] 400 mmol / L HEPES buffer solution (pH 7.5): 104.1 g HEPES, solvent is distilled water.

[0048] Example 1 Construction of recombinant tetrahydrofolate methyltransferase RcoDmdA expression strain and its induced expression

[0049] The tetrahydrofolate methyltransferase RcoDmdA, derived from *Ruegeria conchae*, was retrieved from the GenBank database (GenBank accession No. UWR04622), and its amino acid sequence is shown in SEQ ID NO. 2. After codon optimization, the encoding gene sequence of RcoDmdA was obtained, as shown in SEQ ID NO. 1. This gene was submitted to Genewiz for gene synthesis and cloned into the pET28a plasmid to obtain the recombinant expression plasmid pET28a-RcoDmdA. This plasmid was transformed into the expression host strain *Escherichia coli* BL21(DE3) to obtain the recombinant strain *E. col BL21(DE3)(pET28a-RcoDmdA)*.

[0050] After streaking the recombinant bacteria onto LB agar plates from preserved glycerol tubes and culturing overnight, a single colony was inoculated into LB liquid medium containing 50 μg / mL kanamycin and incubated overnight at 37°C and 200 rpm. The culture was then inoculated into fermentation medium at a rate of 1-2% (v / v) and incubated at 37°C for 2 h. IPTG was added to a final concentration of 0.5 mmol / L, the culture temperature was adjusted to 24°C, and fermentation continued for 10 h to obtain a bacterial agent overexpressing RcoDmdA.

[0051] Example 2: Design and construction of a single-point mutation site for RcoDmdA.

[0052] To improve the catalytic reaction rate and product yield of RcoDmdA, homologous sequence alignment was used to screen for mutation sites. Three-dimensional structural comparison analysis was performed with homologous enzymes of known crystal structures, specifically the crystal structure of PubDmdA derived from *Pelagibacter ubique*, including PDB IDs 3TFH, 3TFI, and 3TFJ. Comparative analysis of the two enzyme domains revealed nine different amino acid residues around the binding pockets of substrates THF and DMSP, including sites 29, 60, 121, 150, 196, 245, 246, 249, and 263. Site-directed substitution mutations were performed at these sites. Based on the gene sequence of wild-type RcoDmdA shown in SEQ ID NO.1, site-directed mutagenesis primers were designed, as shown in Table 1.

[0053] Each site on RcoDmdA is mutated to an amino acid residue corresponding to PubDmdA. The method for constructing site-directed mutants is as follows: using the vector pET28a-RcoDmdA as a template, PCR amplification is performed, and the target mutation site is introduced using primers. After purifying the amplified product using a PCR product purification kit, the purified PCR product is digested with DpnI. The processed PCR product is then ligated using the ClonExpress one-step cloning kit from Novizumi. TM II. After the ligation reaction, the cells were transformed into Escherichia coli BL21(DE3) cells, plated on LB plates containing 50 μg / mL kanamycin, and verified by colony PCR and sequencing to obtain site-directed mutants at 9 sites.

[0054] Table 1 Primers used for site-directed substitution mutations

[0055]

[0056]

[0057] Example 3: Screening and activity comparison analysis of single-point mutants

[0058] The transformants obtained from the plates in Example 2 were transferred to LB liquid medium containing 50 μg / mL kanamycin and cultured on a shaker until mid-log growth. Then, they were transferred to fermentation medium at an inoculum of 1-2% (v / v) and cultured at 37°C for 2 hours. IPTG was added to a final concentration of 0.5 mmol / L, and the fermentation temperature was maintained at 24°C for another 10 hours to obtain the induced bacterial agent.

[0059] Following the method described in Example 1, bacterial agents containing the RcoDmdA mutant were prepared. 50 mL of each agent was centrifuged at 10000×g for 10 min to collect cells. The cells were resuspended in 10 mL of 0.4 mol / L HEPES buffer (pH 7.5) and sonicated. The supernatant was collected by centrifugation. A final concentration of 10.0 g / L THF, 100.0 mmol / L MSDS, and 1.0 g / L DTT were added. The reaction was carried out in a magnetically stirred water bath at 37°C for 10 h. The reaction solution was then used for HPLC analysis.

[0060] The enzyme activity screening results of single-point mutants at various time points are as follows: Figure 9 As shown.

[0061] The catalytic results of the nine single-point mutants are shown in Table 3. By comparing the substrate conversion rates, the preferred mutants F245Y, V121I and F263Y were obtained.

[0062] Table 3 Screening results of single-point mutants

[0063]

[0064] Example 4: Design of RcoDmdA mutation sites and construction of small, efficient mutant libraries

[0065] To further optimize the catalytic pocket of RcoDmdA and thus improve its catalytic reaction rate and product yield, a three-codon saturation mutagenesis was performed on the THF-binding domain of RcoDmdA. Mutation sites were screened using computer-aided design methods, specifically, based on the reported crystal structure of PubDmdA from *Taiwanella spp.* (PDB ID: 3TFH), homology modeling of RcoDmdA was performed using molecular docking software. Considering the THF docking results of RcoDmdA with the substrate, the characteristics of the enzyme's substrate-binding pocket, and the enzyme's catalytic mechanism, 13 amino acid residues were finally identified, specifically positions 29, 93, 107, 121, 122, 150, 175, 176, 193, 194, 242, 243, and 263 of the amino acid sequence SEQ ID NO:2. The specific locations are shown in the figure. Figure 3 As shown, the specific groupings are as follows: Figure 4As shown (different colors represent different groups: green: group A; orange: group B; blue: group C; purple: group D), they are divided into group A (Y29, Y93, P107), group B (V121, A122, F175, F176), group C (P150, S193, G194) and group D (L242, L243, F263).

[0066] Based on the wild-type RcoDmdA gene sequence shown in SEQ ID NO.1, three-codon saturation mutant primers in groups A, B, C, and D were designed, as shown in Table 2. The three amino acids corresponding to the three codons are serine, isoleucine, and tyrosine, respectively. The construction method of the three-codon saturation mutant library is as follows: First, the fragment at the mutation site was amplified by PCR, and the mutation was randomly introduced by primers and purified by gel extraction; then, using one of the purified PCR products as primers, the entire plasmid was further amplified using the recombinant plasmid pET28a-RcoDmdA as a template, and the amplified band of the correct size was obtained by 1% agarose gel electrophoresis. After digesting the PCR amplified product with the restriction endonuclease DpnI, the PCR product was ligated by a one-step cloning method, and then transformed into E. coli BL21(DE3) cells and plated on LB plates with 50 μg / mL kanamycin to obtain the three-codon saturation mutant library.

[0067] Table 2 Primers for triple codon saturation mutations

[0068]

[0069]

[0070]

[0071] Example 5: Screening of four small and precise mutant libraries

[0072] The triple-codon saturated mutant library obtained in Example 4, i.e., the resistant transformants grown on the plate, was transferred to LB liquid medium containing 50 μg / ml kanamycin and cultured on a shaker until mid-log growth. Then, it was transferred to fermentation medium at an inoculum rate of 1-2% (v / v) and cultured at 37°C for 2 hours. IPTG was added to a final concentration of 0.5 mmol / L, and the fermentation temperature was controlled at 24°C for another 10 hours to obtain the induced bacterial agent.

[0073] Take 2 mL of the cultured bacterial agent from each transformant, centrifuge at 10000×g for 5 min to collect the cells, resuspend the cells in 1 mL of 0.4 mol / L HEPES buffer at pH 7.5, add 5.0 g / L THF and 50 mmol / L MSDS, and catalyze the reaction by shaking in a metal bath at 37℃ and 1000 rpm for 2 h. The catalytic reaction solution is used for HPLC analysis.

[0074] The enzyme activity screening results of mutants in libraries LibA, LibB, LibC, and LibD are as follows: Figure 6 , Figure 7 , Figure 8 , Figure 9 As shown.

[0075] By comparing substrate conversion rates, superior mutant strains were selected for sequencing analysis. The results showed that mutant strains F263Y, V121I, P107I, V121S, A122S, and F263I were the preferred mutants obtained from the first-generation library screening.

[0076] Example 6: Activity analysis of the first-generation preferred mutant catalyzing the preparation of L-5-MTHF from THF

[0077] Following the method described in Example 1, bacterial agents containing RcoDmdA mutants F263Y, V121I, V121S, A122S, F263I, and P107I were prepared. 30 mL of each agent was centrifuged at 10000×g for 10 min to collect cells, which were then resuspended in 10 mL of 0.4 mol / L HEPES buffer (pH 7.5). A final concentration of 20.0 g / L THF, 100.0 mmol / L MSDS, and 1.0 g / L LTT were added. The reaction was carried out in a magnetically stirred water bath at 37°C for 10 h. The resulting reaction solution was then used for HPLC analysis.

[0078] The catalytic results of the first-generation preferred mutants are shown in Table 4. Among them, mutant strain F263Y showed the best catalytic activity against the substrate THF and was identified as the first-generation optimal mutant.

[0079] Table 4. Results of secondary screening of first-generation preferred mutants

[0080]

[0081]

[0082] Example 7: Application of the superior mutant RcoDmdA-F263Y in the preparation of L-5-MTHF from THF catalyzed by it.

[0083] Induced expression of the RcoDmdA mutant strain. Recombinant strain E. coli BL21(DE3)(pET28a-RcoDmdA-F263Y) was streaked onto LB agar plates from preserved glycerol tubes and cultured overnight. Single colonies were then inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 200 rpm. Seed culture was then inoculated at a rate of 2% into LB medium containing kanamycin sulfate (50 μg / mL) and cultured at 37°C and 200 rpm for 4–6 h on a shaker. Afterward, a 2% inoculation was carried out in a 3L fermenter and fermented at 37°C. When OD... 600 When the concentration reaches approximately 10, 10 g / L lactose is added to induce the expression of the target protein. The induction temperature is 22℃. After continuing the induction culture for another 8 hours, the fermentation is terminated, and the fermentation broth inoculum for the catalytic reaction is obtained.

[0084] The catalytic reaction was carried out with a 1 g / L LTHF feed. After centrifugation to collect the bacterial cells, they were resuspended in 0.4 mol / L HEPES buffer (pH 7.5) to prepare cell density OD. 600 The concentration was 40. After high-pressure homogenization, the cell lysate was 30 mL, which was poured into a round-bottom flask and incubated in a 37°C water bath for 10 min. Then, 1 g / L THF, 20 mmol / L MSDS, and 1.0 g / L LTT were added sequentially. Magnetic stirring was immediately started to mix thoroughly, and the reaction start time was recorded. After 1 h of catalytic reaction, the L-5-MTHF yield was 1.052 g / L, and the conversion rate reached 102%.

[0085] The catalytic reaction was carried out with a feed of 10 g / L THF. After centrifugation to collect the bacterial cells, they were resuspended in 0.4 mol / L HEPES buffer (pH 7.5) to prepare cell density OD. 600 The concentration was 40. After high-pressure homogenization, the cell lysate was 30 mL, which was poured into a round-bottom flask and incubated in a 37°C water bath for 10 min. Then, 10 g / L THF, 100 mmol / L MSDS, and 1.0 g / L LTT were added sequentially. The magnetic stirrer was immediately turned on to mix thoroughly. The start of the reaction was recorded and the time was started. After 5 h of catalytic reaction, the yield of L-5-MTHF was 9.9 g / L, and the conversion rate reached 99%.

[0086] The catalytic reaction was carried out with a feed of 15 g / L LTHF. After centrifugation to collect the bacterial cells, they were resuspended in 0.4 mol / L HEPES buffer (pH 7.5) to prepare cell density OD. 600The concentration was 40. After high-pressure homogenization, the cell lysate was 30 mL, which was poured into a round-bottom flask and incubated in a 37°C water bath for 10 min. Then, 15 g / L THF, 150 mmol / L MSDS, and 1.0 g / L LTT were added sequentially. Magnetic stirring was immediately started to mix thoroughly, and the reaction start time was recorded. After 10 h of catalytic reaction, the L-5-MTHF yield was 14.9 g / L, and the conversion rate reached 99.4%.

[0087] The catalytic reaction was initiated with a 20 g / L THF feed. After centrifugation to collect the bacterial cells, they were resuspended in 0.4 mol / L HEPES buffer (pH 7.5) to prepare cell density OD. 600 The concentration was 40. After high-pressure homogenization, the cell lysate was 30 mL, which was poured into a round-bottom flask and incubated in a 37°C water bath for 10 min. Then, 20 g / L THF, 150 mmol / L MSDS, and 1.0 g / L LTT were added sequentially. Magnetic stirring was immediately started to mix thoroughly, and the reaction start time was recorded. After 12 hours of catalytic reaction, the yield of L-5-MTHF was 17.1 g / L, and the conversion rate reached 85%.

[0088] The catalytic reaction was carried out with a feed of 30 g / L LTHF. After centrifugation to collect the bacterial cells, they were resuspended in 0.4 mol / L HEPES buffer (pH 7.5) to prepare cell density OD. 600 The concentration was 40. After high-pressure homogenization, the cell lysate was 30 mL, which was then poured into a round-bottom flask and incubated in a 37°C water bath for 10 min. Then, 30 g / L THF, 300 mmol / L MSDS, and 1.0 g / L DTT were added sequentially. Magnetic stirring was immediately started to mix thoroughly, and the reaction start time was recorded. The conversion rates at 8 h and 13 h were 55.2% and 63.3%, respectively.

Claims

1. A tetrahydrofolate methyltransferase RcoDmdA mutant, characterized in that: The mutant amino acid sequence is shown as SEQ ID NO.

3.

2. A gene encoding the tetrahydrofolate methyltransferase RcoDmdA mutant of claim 1.

3. The gene of claim 2, wherein: The encoding gene nucleotide sequence is shown as SEQ ID NO.

10.

4. A recombinant vector containing the gene of claim 3.

5. A genetically engineered bacterium containing the gene of claim 3.

6. Use of the tetrahydrofolate methyltransferase RcoDmdA mutant of claim 1 in enzyme-catalyzed preparation of L-5-methyltetrahydrofolate from tetrahydrofolate (THF).

7. Use according to claim 6, wherein: The application constructs a genetically engineered bacterium containing the coding gene of the mutant, and uses the wet bacterium or enzyme-containing cells obtained by crushing the bacterium body of the genetically engineered bacterium cultured by fermentation as a catalyst to methylate THF to obtain L -5-MTHF.

8. Use according to claim 7, wherein: The recombinant bacteria were constructed by using pET28a plasmid and E. coli BL21 host cells E. coli BL21.

9. Use according to claim 7 or 8, characterized in that: The methylation is carried out under the condition of adding a methyl donor dimethyl sulfonium chloride.

10. Use according to claim 7 or 8, characterized in that: The methylation reaction has a pH value of 6.0-9.0 and a reaction temperature of 25-50°C, and the product is obtained L -5-MTHF.

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