Tetrahydrofolate methyltransferase mutant, gene encoding same and use thereof

Abstract

A tetrahydrofolate methyltransferase mutant, a gene encoding same and the use thereof. Provided is a tetrahydrofolate methyltransferase RcoDmdA mutant, which has an amino acid sequence as represented by SEQ ID NO. 3. The RcoDmdA mutant has higher catalytic activity. The substrate THF feeding amount is significantly increased to 15-20 g / L, the yield of product L-5-MTHF is increased to 15-17 g / L, and the substrate conversion rate is high. The tetrahydrofolate methyltransferase mutant and the gene encoding same have great value for industrial application.

Description

A tetrahydrofolate methyltransferase mutant, its encoding gene and its application Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering, and specifically relates to a tetrahydrofolate methyltransferase mutant, its encoding gene, and its applications. Background Technology

[0002] L-5-Methyltetrahydrofolate (L-5-MTHF) is the natural active form of folic acid (FA) and the main folic acid component in the circulatory system, and it can be directly absorbed and utilized. It is currently recommended as a substitute for synthetic folic acid. The advantage of L-5-MTHF as a nutritional supplement lies in its higher bioavailability; it can be directly absorbed and utilized without metabolic conversion, thus avoiding the problem of insufficient absorption and utilization of synthetic folic acid caused by DHFR and MTHFR gene polymorphisms. Active folic acid L-5-MTHF possesses the properties of natural folic acid and does not lead to the accumulation of unmetabolized synthetic folic acid in the blood.

[0003] Due to its high bioavailability, L-5-MTHF's stable structure, L-5-methyltetrahydrofolate calcium, was approved by the US FDA in 2001 and used 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 folic acid and is widely used in the pharmaceutical, food, and livestock industries. Existing commercial L-5-MTHF products are prepared chemically, starting with synthetic folic acid, chemically hydrogenating it to obtain tetrahydrofolate, then resolving it through crystallization to obtain (6S,αS)tetrahydrofolate, followed by further methylation and reduction reactions to obtain the physiologically active (6S,αS)-5-MTHF (L-5-MTHF). This reaction process is complex, costly, and contains impurities such as the physiologically inactive (6R,αS)-5-MTHF.

[0004] With the rapid development of biotechnology, green, environmentally friendly, efficient, and low-cost production methods have gradually attracted attention. Among them, the green synthesis technology of L-5-MTHF using bio-enzymatic catalysis is being continuously explored and improved, including bio-fermentation (Microb. Biotechnol. 2022, 15:2758-2772, J. Agric. Food. Chem. 2022, 70:5849-5859) and enzyme catalysis (CN116574768A). Bio-enzymatic methods have the characteristics of high catalytic efficiency, high selectivity, mild reaction conditions, low energy consumption, and non-toxicity, which are in line with the green development direction. Therefore, bio-enzymatic preparation processes based on highly active bioenzymes have important application value.

[0005] A tetrahydrofolate methyltransferase mutant derived from Ruegeria conchae in the Indian Ocean has been disclosed. It can catalyze the efficient methylation of tetrahydrofolate (THF) to L-5-MTHF and features high substrate feed rate and fast catalytic reaction rate. Summary of the Invention

[0006] To overcome the problems of low catalytic efficiency, low yield, and low conversion rate in existing technologies, this invention provides a tetrahydrofolate methyltransferase mutant, its encoding gene, and its applications.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a tetrahydrofolate methyltransferase RcoDmdA mutant, wherein the amino acid sequence of the mutant is shown in one of SEQ ID NO.3 to 9.

[0008] This invention relates to a mutated tetrahydrofolate methyltransferase RcoDmdA derived from Ruegeria conchae in the Indian Ocean, which enhances its catalytic activity. The amino acid sequence of RcoDmdA is shown in SEQ ID NO.2.

[0009] This invention was obtained by mutating amino acid residue sites in the methyl acceptor substrate THF binding domain of RcoDmdA, including sites V121, P150, S196, F245, M249, and F263; and amino acid sites in the methyl donor substrate dimethyl mercaptopropionic acid (DMSP) binding domain, including sites Y29, C60, and G249.

[0010] This invention utilizes computer-aided software to perform homology modeling of wild-type RcoDmdA and compares it with known homologous crystal structures, namely tetrahydrofolate methyltransferases PubDmdA (PDB ID: 3TFI and 3TFJ) derived from Pelagibacter ubique. Key amino acid residue sites are identified and screened, as shown in Figure 2. Mutant enzymes are obtained through site-directed substitution mutations, and superior mutants are screened.

[0011] This invention relates to the THF binding pocket around RcoDmdA. The 13 amino acid residues within the range, including Y29, Y93, P107, V121, A122, P150, F175, F176, S193, G194, L242, L243, and F263, were screened and obtained as superior mutants by constructing a small and precise combined mutant library, namely the triple codon saturation mutagenesis technique.

[0012] 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 constructs four small and efficient libraries according to the scheme shown in Figure 4. The three selected 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.

[0013] The amino acid sequences of the mutants described in this invention are shown in SEQ ID NO. 3 to 9, which are RcoDmdA-F263Y, RcoDmdA-V121I, RcoDmdA-A122S, RcoDmdA-V121S, RcoDmdA-P107I, RcoDmdA-F263I, and RcoDmdA-F245Y, respectively.

[0014] Secondly, the present invention provides a gene for a tetrahydrofolate methyltransferase RcoDmdA mutant.

[0015] Preferably, the nucleotide sequence of the encoding gene is shown in any one of SEQ ID NO. 10 to 16, encoding RcoDmdA-F263Y, RcoDmdA-V121I, RcoDmdA-A122S, RcoDmdA-V121S, RcoDmdA-P107I, RcoDmdA-F263I, and RcoDmdA-F245Y, respectively.

[0016] The coding gene of the superior mutant F263Y described in this invention can be synthesized by codon optimization based on its amino acid sequence. Preferably, the nucleotide sequence of the coding gene is shown in SEQ ID NO.10.

[0017] Thirdly, the present invention also provides a recombinant vector for the encoded gene.

[0018] Fourthly, the present invention also relates to genetically engineered bacteria that encode the aforementioned gene.

[0019] 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).

[0020] 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.

[0021] 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.

[0022] Preferably, the pET28a plasmid and Escherichia coli BL21 host cells are used to construct recombinant E. coli BL21.

[0023] 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).

[0024] Preferably, the catalytic activity is carried out under conditions of adding the methyl donor dimethyl sulfonium chloride.

[0025] 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).

[0026] 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.

[0027] 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%.

[0028] The beneficial effects of the present invention are mainly reflected in: (1) The RcoDmdA mutant provided by the present invention has higher catalytic activity; (2) The substrate THF feed amount is significantly increased to 15-20 g / L, the product L-5-MTHF yield is increased to 15-17 g / L, and the substrate conversion rate is high; (3) The tetrahydrofolate methyltransferase mutant and the encoding gene provided by the present invention have important industrial application value. Attached Figure Description

[0029] Figure 1 is a schematic diagram of the structure of wild-type tetrahydrofolate methyltransferase RcoDmdA.

[0030] Figure 2 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.

[0031] Figure 3 shows the interaction between wild-type tetrahydrofolate methyltransferase RcoDmdA and THF. A schematic diagram of the 13 unsaturated amino acid sites within the range.

[0032] Figure 4 is a schematic diagram of the grouping and construction of small and precise libraries with three-codon saturation mutations.

[0033] Figure 5 is a schematic diagram of the initial screening results of the A group mutant library in the first-generation three-codon saturated mutant library.

[0034] Figure 6 is a schematic diagram of the initial screening results of the B group mutant library in the first-generation three-codon saturated mutant library.

[0035] Figure 7 is a schematic diagram of the initial screening results of the C group mutant library in the first-generation three-codon saturated mutant library.

[0036] Figure 8 is a schematic diagram of the initial screening results of the D group mutant library in the first-generation three-codon saturated mutant library.

[0037] Figure 9 shows a comparison of the product synthesis curves for the preparation of L-5-MTHF from THF catalyzed by a single-point mutant.

[0038] Figure 10 shows the HPLC chromatograms of THF and L-5-MTHF standard samples. Detailed Implementation

[0039] 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.

[0040] 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.

[0041] LB medium: yeast extract 5.0 g / L, peptone 10.0 g / L, NaCl 10.0 g / L, solvent is distilled water.

[0042] 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.

[0043] 400 mmol / L HEPES buffer solution (pH 7.5): 104.1 g HEPES, solvent: distilled water. Example 1: Construction and induction of recombinant tetrahydrofolate methyltransferase RcoDmdA expression strain.

[0044] 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 RcoDmdA coding gene sequence 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)*.

[0045] 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 hours. 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 hours to obtain a bacterial agent overexpressing RcoDmdA. Example 2: Design and construction of a single-point mutation site for RcoDmdA.

[0046] 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.

[0047] 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. Following the ligation reaction, the mutants were transformed into *E. coli* BL21(DE3) cells and plated on LB agar plates containing 50 μg / mL kanamycin. Verification was performed by colony PCR and sequencing to obtain site-directed mutants at nine sites. Table 1 shows the primers used for site-directed substitution mutations. Example 3: Screening and activity comparison analysis of single-point mutants

[0048] 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.

[0049] 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 dimethylsulfonium chloride (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.

[0050] Figure 9 shows the enzyme activity screening results of single-point mutants at various time points.

[0051] 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. Table 3: Screening results of single-point mutants. Example 4: Design of RcoDmdA mutation sites and construction of small, efficient mutant libraries

[0052] 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 *Oceanophora glomerata* (PDB ID: 3TFH), homology modeling of RcoDmdA was performed using molecular docking software. Considering the docking results of RcoDmdA with the substrate THF, 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 Figure 3, and the specific groupings are shown in Figure 4 (different colors represent different groups: green: group A; orange: group B; blue: group C; purple: group D). These groups 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).

[0053] Based on the wild-type RcoDmdA gene sequence shown in SEQ ID NO.1, three-codon saturation mutation primers (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 mutation library is as follows: First, the fragment at the mutation site was amplified by PCR, and the mutation was randomly introduced using primers and purified by gel extraction. Then, using one of the purified PCR products as primers, the entire plasmid pET28a-RcoDmdA was further amplified using the recombinant plasmid pET28a-RcoDmdA as a template. 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 using a one-step cloning method, and then transformed into E. coli BL21(DE3) cells and plated on LB plates containing 50 μg / mL kanamycin to obtain the three-codon saturation mutation library. Table 2 Primers used for three-codon saturation mutation. Example 5: Screening of four small and precise mutant libraries

[0054] 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.

[0055] 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 in a metal bath at 37℃ and 1000 rpm for 2 h. The catalytic reaction solution is used for HPLC analysis.

[0056] The enzyme activity screening results of mutants in libraries LibA, LibB, LibC, and LibD are shown in Figures 6, 7, 8, and 9, respectively.

[0057] By comparing substrate conversion rates, superior mutant strains were selected for sequencing analysis. The results showed that mutants F263Y, V121I, P107I, V121S, A122S, and F263I were the preferred mutants obtained from the first-generation library screening. Example 6: Activity analysis of the first-generation preferred mutants catalyzing the preparation of L-5-MTHF from THF.

[0058] Following the method described in Example 1, bacterial agents containing the 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 DTT 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.

[0059] The catalytic results of the first-generation selected mutants are shown in Table 4. Among them, mutant F263Y showed the best catalytic activity against the substrate THF and was identified as the optimal mutant of the first generation. Table 4: Rescreening results of the first-generation selected mutants. Example 7 Application of the superior mutant RcoDmdA-F263Y in the preparation of L-5-MTHF from THF catalyzed by THF

[0060] 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.

[0061] Catalytic reaction with 1 g / L THF feed. After centrifugation to collect 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%.

[0062] Catalytic reaction catalyzed by 10 g / L THF feed. After centrifugation to collect 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 DTT 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%.

[0063] Catalytic reaction catalyzed by 15 g / L THF feed. After centrifugation to collect 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, 15 g / L THF, 150 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. After 10 h of catalytic reaction, the yield of L-5-MTHF was 14.9 g / L, and the conversion rate reached 99.4%.

[0064] Catalytic reaction catalyzed by 20 g / L THF feed. After centrifugation to collect 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 DTT 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 L-5-MTHF yield was 17.1 g / L, and the conversion rate reached 85%.

[0065] Catalytic reaction catalyzed by 30 g / L THF feed. After centrifugation to collect 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, 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 amino acid sequence of the mutant is shown in SEQ ID NO.

3.

2. The gene encoding a tetrahydrofolate methyltransferase RcoDmdA mutant as described in claim 1.

3. The genetic code of claim 2, wherein: The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

10.

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

5. Genetically engineered bacteria containing the encoding gene of claim 3.

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

7. Use according to claim 6, wherein: The application involves constructing a genetically engineered bacterium containing the mutant encoding gene, and using 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.

8. The application as described in claim 7, characterized in that: Recombinant E. coli BL21 was constructed using pET28a plasmid and E. coli BL21 host cells.

9. The application as described in claim 7 or 8, characterized in that: The catalytic activity was carried out under conditions of adding the methyl donor dimethyl sulfonium chloride.

10. The application as described in claim 7 or 8, characterized in that: The catalytic reaction has a pH value of 6.0-9.0 and a reaction temperature of 25℃-50℃.

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