Tetrahydrofolate methyltransferase mutant, coding gene and application of tetrahydrofolate methyltransferase mutant

Abstract

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

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and enzyme engineering, and specifically relates to a tetrahydrofolate methyltransferase mutant, a coding gene and an application thereof. Background Art

[0002] L-5-Methyltetrahydrofolate (L-5-MTHF) is the natural active form of folic acid (FA) and the main component of folic acid in the blood circulation system. It can be directly absorbed and utilized. It has been suggested 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 problem of insufficient absorption and utilization of synthetic folic acid caused by DHFR and MTHFR gene polymorphisms. The active folic acid L-5-MTHF has the properties of natural folic acid and will not cause the problem of unmetabolized synthetic folic acid accumulating in the blood.

[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 used as a new raw material in nutritional health products. It was then used in the international market as a safer folic acid food additive. At present, 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. The existing L-5-MTHF products are prepared by chemical methods, that is, synthetic folic acid is used as the starting material, and the unsaturated tetrahydrofolate is prepared by chemical hydrogenation, and then (6S, αS) tetrahydrofolate is obtained by crystallization and splitting, and further methylation and reduction reactions are performed to obtain physiologically active (6S, αS)-5-MTHF (L-5-MTHF). The reaction process is complicated, the preparation cost is high, and it contains impurity components of (6R, αS)-5-MTHF that is not physiologically active.

[0004] With the rapid development of biotechnology, green, environmentally friendly, efficient and low-cost production methods have gradually attracted people's attention. Among them, the green synthesis technology of L-5-MTHF catalyzed by bioenzyme has also been continuously explored and improved, including biofermentation (Microb. Biotechnol. 2022, 15: 2758-2772, J. Agric. Food. Chem. 2022, 70: 5849-5859) and enzyme catalysis (CN116574768A). The bioenzyme method has the characteristics of high catalytic efficiency, high selectivity, mild reaction conditions, low energy consumption, and non-toxicity, which is in line with the green development direction. Therefore, the bioenzyme preparation process based on high-activity bioenzymes has important application value.

[0005] A tetrahydrofolate methyltransferase mutant from Ruegeria conchae is disclosed, which can catalyze the efficient methylation of tetrahydrofolate (THF) to produce L-5-MTHF and has the characteristics of high substrate input amount and fast catalytic reaction rate. Summary of the invention

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

[0007] In order to achieve the above object, the present invention is implemented by the following technical solutions: 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 NOs. 3-9.

[0008] The present invention performs mutation modification on tetrahydrofolate methyltransferase RcoDmdA derived from Ruegeria conchae in the Indian Ocean to improve its catalytic activity. The amino acid sequence of RcoDmdA is shown in SEQ ID NO.2.

[0009] The invention obtains the RcoDmdA by mutating 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 dimethylmercaptopropionic acid DMSP binding domain, including the Y29, C60 and G249 sites.

[0010] The present invention uses computer-aided software to perform homology modeling on wild-type RcoDmdA and compares it with known homologous crystal structures, namely the tetrahydrofolate methyltransferase PubDmdA from Pelagibacter ubique (PDB ID: 3TFI and 3TFJ), to identify and screen key amino acid residue sites. Figure 2 As shown, mutant enzymes were obtained by site-directed replacement mutagenesis, and excellent mutants were screened.

[0011] The present invention is to study the THF binding pocket of RcoDmdA Thirteen 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 by constructing a small and precise combinatorial mutation library, namely the three-codon saturation mutagenesis technology.

[0012] The present invention divides the above-selected 13 amino acid residues into four groups, namely, group A (Y29, Y93, P107), group B (V121, A122, F175, F176), group C (P150, S193, G194) and group D (L242, L243, F263), and according to the following: Figure 4 The scheme shown constructs four small and fine libraries. The three codons selected are isoleucine, serine and tyrosine, which are used for three-codon saturation mutation at each site. Excellent mutants are obtained by screening each mutant library.

[0013] The amino acid sequences of the excellent mutants of the present 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] In a second aspect, the present invention provides a gene of a tetrahydrofolate methyltransferase RcoDmdA mutant.

[0015] Preferably, the nucleotide sequence of the encoding gene is shown as one of SEQ ID NOs. 10 to 16.

[0016] The coding genes of the excellent mutants F263Y, V121I, A122S, V121S, P107I, F263I and F245Y of the present invention can be synthesized after codon optimization according to their amino acid sequences. Preferably, the nucleotide sequences of the coding genes are shown in SEQ ID NOs. 10 to 16.

[0017] In a third aspect, the present invention also provides a recombinant vector encoding the gene.

[0018] In a fourth aspect, the present invention also relates to a genetically engineered bacterium encoding the gene.

[0019] In a fifth aspect, the present invention also provides the use of the tetrahydrofolate methyltransferase RcoDmdA mutant in the preparation of L-5-methyltetrahydrofolate by enzyme-catalyzed tetrahydrofolate THF.

[0020] Preferably, the application constructs a genetically engineered bacterium containing the mutant encoding gene, and uses wet cells obtained by fermentation culture of the genetically engineered bacterium or enzyme-containing cells obtained by crushing the cells as a catalyst to methylate THF to obtain L-5-MTHF.

[0021] The 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 for catalyzing the conversion of THF into L-5-MTHF.

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

[0023] The expression plasmid and host cell of the present invention are preferably pET28a plasmid and Escherichia coli BL21 (DE3) host cell, that is, 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 catalysis is carried out under the condition of adding dimethylsulfonium chloride as a methyl donor.

[0025] The method of the present invention is characterized in that the wet bacteria or the supernatant of the cell lysis liquid obtained by breaking the bacteria is used to catalyze the conversion of THF into L-5-MTHF, which is characterized in that the cell density OD 600 The molecular weight of the reaction mixture is 20 to 80 (preferably 40), the amount of THF as substrate is 1 g / L to 20 g / L (preferably 10-15 g / L), and the amount of dimethylsulfonium chloride as methyl donor substrate is 10 mM to 300 mM (preferably 150 mM).

[0025] Preferably, the catalytic reaction pH value is 6.0-9.0, the reaction temperature is 25° C.-50° C., and the product L-5-MTHF can be obtained.

[0026] The catalytic reaction pH value of the present invention is 6.0-9.0 (preferably 7.5), the reaction temperature is 25°C-50°C (preferably 37°C), and the product L-5-MTHF can be obtained, and the conversion rate reaches 85%-99%.

[0027] 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 amount of THF substrate fed was significantly increased to 15-20 g / L, the yield of the product L-5-MTHF was increased to 15-17 g / L, and the substrate conversion rate was high; (3) The tetrahydrofolate methyltransferase mutant and encoding gene provided by the present invention have important industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the structure of wild-type tetrahydrofolate methyltransferase RcoDmdA.

[0029] Figure 2 Schematic diagram of the amino acid residue sites around the binding pocket of wild-type tetrahydrofolate methyltransferase RcoDmdA with THF and methyl donor DMSP.

[0030] Figure 3 The wild-type tetrahydrofolate methyltransferase RcoDmdA and THF surrounding Schematic diagram of the 13 unsaturated amino acid sites within the range.

[0031] Figure 4 Schematic diagram for the construction of a small and precise library of three-codon saturation mutations.

[0032] Figure 5 Schematic diagram of the initial screening results of the Group A mutant library in the first-generation three-codon saturation mutation library.

[0033] Figure 6 Schematic diagram of the initial screening results of the Group B mutant library in the first-generation three-codon saturation mutation library.

[0034] Figure 7 Schematic diagram of the initial screening results of the C group mutant library in the first-generation three-codon saturation mutation library.

[0035] Figure 8 Schematic diagram of the initial screening results of the D group mutant library in the first-generation three-codon saturation mutation library.

[0036] Fig. 9 Comparison of product synthesis curves for the preparation of L-5-MTHF from THF catalyzed by single-point mutants.

[0037] Fig.10 HPLC chromatograms of THF and L-5-MTHF standard samples. DETAILED DESCRIPTION

[0038] The following is an explanation of the embodiments of the present invention by specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied by other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. In the embodiments of the present invention, the methods used are all conventional methods unless otherwise specified, and the reagents used can be obtained from commercial sources.

[0039] The present invention will be further described in detail below with reference to specific embodiments. The embodiments are only used to explain the present invention and do not limit the protection scope of the present invention.

[0040] LB medium: yeast powder 5.0 g / L, peptone 10.0 g / L, NaCl 10.0 g / L, and the solvent is distilled water.

[0041] Fermentation medium: yeast powder 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 is distilled water.

[0042] 400mmol / L HEPES buffer solution (pH 7.5): HEPES 104.1g, the solvent is distilled water. Example 1 Construction of recombinant tetrahydrofolate methyltransferase RcoDmdA expression strain and its induced expression

[0043] Tetrahydrofolate methyltransferase RcoDmdA from Ruegeria conchae was retrieved from the GenBank database, i.e., GenBank accession No. UWR04622, and its amino acid sequence is shown in SEQ ID NO. 2. After codon optimization, the RcoDmdA encoding gene sequence was obtained, as shown in SEQ ID NO. 1. The gene was submitted to Jin Weizhi Company for gene synthesis and cloned into the pET28a plasmid to obtain the recombinant expression plasmid pET28a-RcoDmdA. The plasmid was transformed into the expression host Escherichia coli BL21 (DE3) strain to obtain the recombinant strain E.col BL21 (DE3) (pET28a-RcoDmdA).

[0044] After streaking the recombinant bacteria from the preserved glycerol tube on an LB plate for overnight culture, a single colony was inoculated into a LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C and 200 rpm. The inoculation amount was inoculated into a fermentation medium at 1-2% (v / v), and cultured at 37°C for 2 hours; IPTG was added at a final concentration of 0.5 mmol / L, the culture temperature was adjusted to 24°C, and the fermentation was continued for 10 hours to obtain a bacterial agent that overexpressed RcoDmdA. Example 2 Design of single point mutation site of RcoDmdA and construction of mutation site

[0045] In order to improve the catalytic reaction rate and product yield of RcoDmdA, the homologous sequence alignment method was used to screen mutation sites. The three-dimensional structure was compared and analyzed with homologous enzymes with known crystal structures, namely the crystal structures of PubDmdA from Pelagibacter ubique, including PDB IDs 3TFH, 3TFI and 3TFJ. Through comparative analysis of the structural domains of the two enzymes, it was found that there were 9 different amino acid residues around the substrate THF and substrate DMSP binding pockets, including sites 29, 60, 121, 150, 196, 245, 246, 249 and 263, and these sites were subjected to site-directed substitution mutation. According to the gene sequence of the wild-type RcoDmdA shown in SEQ ID NO.1, site-directed mutagenesis primers were designed, as shown in Table 1.

[0046] Each site on RcoDmdA mutates to an amino acid residue corresponding to PubDmdA. The method for constructing site-directed mutants is as follows: using vector pET28a-RcoDmdA as a template, PCR amplification is performed, and the target mutation site is introduced through primers. After the amplified product is purified using a PCR product purification kit, the purified PCR product is digested with DpnⅠ. The treated PCR product is subjected to a ligation reaction, that is, the one-step cloning kit ClonExpress from Novazon is used. TM II. After the ligation reaction, the cells were transformed into E. coli BL21 (DE3) cells, coated on LB plates containing 50 μg / mL of kanamycin, and verified by colony PCR and sequencing, thereby obtaining site-directed mutants at 9 sites. Table 1 Primers used for site-directed substitution mutagenesis Example 3 Screening of single point mutants and comparative analysis of their activity

[0047] The transformants on the plate obtained in Example 2 were transferred to LB liquid medium containing 50 μg / mL kanamycin and cultured on a shaking platform until the mid-logarithmic growth period. Then, the inoculation amount was transferred to the fermentation medium at 1-2% (v / v), cultured at 37°C for 2 hours; IPTG with a final concentration of 0.5 mmol / L was added, the fermentation temperature was controlled at 24°C, and the fermentation was continued for 10 hours to obtain the bacterial agent after induced expression.

[0048] According to the method described in Example 1, the bacterial agents containing RcoDmdA mutants were prepared. 50 mL of each bacterial agent was centrifuged at 10000×g for 10 min to collect cells, and the cells were resuspended in 10 mL of 0.4 mol / L HEPES buffer at pH 7.5 for ultrasonic disruption, and the supernatant was centrifuged. THF, 100.0 mmol / L dimethylsulfonium chloride (MSDS), and 1.0 g / L DTT were added at a final concentration of 10.0 g / L. After catalytic reaction in a 37°C magnetic stirring water bath for 10 hours, the catalytic reaction solution was used for HPLC analysis.

[0049] The results of enzyme activity screening of single point mutants at different time periods are shown in Figure 2. Fig. 9 shown.

[0050] 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 a small and precise mutation library

[0051] In order to further optimize the catalytic pocket of RcoDmdA, thereby improving its catalytic reaction rate and product yield, three codon saturation mutations were performed on the THF binding domain of RcoDmdA. The mutation sites were screened using a computer-aided design method, that is, based on the reported crystal structure of PubDmdA from Bacillus thuringiensis (PDB ID: 3TFH), homology modeling of RcoDmdA was performed using molecular docking software. Taking into account the docking results of RcoDmdA and the substrate THF, the characteristics of the enzyme's substrate binding pocket, and the catalytic mechanism of the enzyme, 13 amino acid residue sites were finally determined, namely the 29th, 93rd, 107th, 121st, 122nd, 150th, 175th, 176th, 193rd, 194th, 242nd, 243rd and 263rd sites of the amino acid sequence SEQ ID NO:2. The specific positions are as follows: Figure 3 The specific groups are as shown in Figure 4As shown (different colors represent different groups, green: group A; orange: group B; blue: group C; purple: group D), and they were divided into group A (Y29, Y93, P107), group B (V121, A122, F175, F176), group C (P150, S193, G194) and group D (L242, L243, F263).

[0052] According to the gene sequence of wild-type RcoDmdA shown in SEQ ID NO.1, four groups of three-codon saturation mutation primers 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 method for constructing a three-codon saturation mutation library is as follows: first, the fragment of the mutation site is PCR amplified, mutations are randomly introduced by primers, and gel recovery and purification are performed; then, the purified PCR product is used as one of the primers, and the recombinant plasmid pET28a-RcoDmdA is used as a template to further amplify the entire plasmid, and the amplified band of the correct size is obtained by 1% agarose gel electrophoresis detection. After the PCR amplification product is digested with the restriction endonuclease DpnI, the PCR product is subjected to a one-step cloning method for ligation reaction, and then transformed into Escherichia coli BL21 (DE3) cells, and coated on an LB plate with 50 μg / mL kanamycin, thereby obtaining a three-codon saturation mutation library. Table 2 Primers used for three-codon saturation mutagenesis Example 5 Screening of four small and fine mutant libraries

[0053] The three-codon saturation mutation library obtained in Example 4, i.e., the resistant transformants grown on the plate were picked and transferred to LB liquid medium containing 50 μg / ml kanamycin, and cultured on a shaking platform until the mid-logarithmic growth period. Then, the inoculum was transferred to the fermentation medium at a rate of 1-2% (v / v), and cultured at 37°C for 2h; IPTG was added at a final concentration of 0.5 mmol / L, and the fermentation temperature was controlled at 24°C, and the fermentation was continued for 10h to obtain the bacterial agent after induced expression.

[0054] Take 2 mL of the culture medium of each transformant, collect the cells by centrifugation at 10000×g for 5 min, 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 in a metal bath at 37°C and 1000 rpm for 2 h. The catalytic reaction liquid is used for HPLC analysis.

[0055] The results of enzyme activity screening of mutants in library LibA, library LibB, library LibC and library LibD are as follows: Figure 6 , Figure 7 , Figure 8 , Fig. 9 shown.

[0056] By comparing the substrate conversion rates, excellent mutants were selected for sequencing analysis, and the results showed that mutants F263Y, V121I, P107I, V121S, A122S, and F263I were the preferred mutants obtained by screening the first-generation library. Example 6 Analysis of the activity of the first generation preferred mutants in catalyzing the preparation of L-5-MTHF from THF

[0057] The bacterial preparations of RcoDmdA mutants F263Y, V121I, V121S, A122S, F263I, and P107I were prepared according to the method described in Example 1. 30 mL of each bacterial preparation was centrifuged at 10000×g for 10 min to collect cells, and the cells were resuspended in 10 mL of 0.4 mol / L HEPES buffer (pH 7.5). THF, 100.0 mmol / L MSDS, and 1.0 g / L DTT were added at a final concentration of 20.0 g / L. After catalytic reaction in a 37°C magnetic stirring water bath for 10 hours, the catalytic reaction solution was used for HPLC analysis.

[0058] The catalytic results of the first generation preferred mutants are shown in Table 4, among which the mutant F263Y has the best catalytic activity for substrate THF and is identified as the first generation optimal mutant. Table 4 Results of rescreening of the first generation of preferred mutants Example 7 Application of the superior mutant RcoDmdA-F263Y in catalyzing the preparation of L-5-MTHF from THF

[0059] Inducible expression of RcoDmdA mutant strain. The recombinant strain E. coli BL21 (DE3) (pET28a-RcoDmdA-F263Y) was streaked from the stored glycerol tube on an LB plate for overnight culture, and then a single colony was inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C and 200 rpm. The seed liquid was inoculated into LB medium containing kanamycin sulfate (50 μg / mL) at a 2% inoculation rate, and cultured in a shaker at 37°C and 200 rpm for 4 to 6 hours. After that, it was inoculated into a 3L fermenter at a 2% inoculation rate and fermented at 37°C. When OD 600When the pH value reached about 10, 10 g / L lactose was added to induce the expression of the target protein. The induction temperature was 22°C. After continuing the induction culture for 8 hours, the fermentation was terminated to obtain the fermentation broth bacterial agent used for catalytic reaction.

[0060] After centrifugation, the cells were collected and resuspended in 0.4 mol / L HEPES buffer (pH 7.5) to prepare a cell density OD 600 The cell lysate was 40, and 30 mL of the cell lysate was obtained after high pressure homogenization. The lysate was poured into a round-bottom flask and incubated in a 37°C constant temperature water bath for 10 min. Then 1 g / L THF, 20 mmol / L MSDS and 1.0 g / L DTT were added successively. Immediately turn on the magnetic stirring to mix evenly, record the start of the reaction and time it. When the catalytic reaction liquid was 1 hour old, the yield of L-5-MTHF was 1.052 g / L, and the conversion rate reached 102%.

[0061] The catalytic reaction was carried out with 10 g / L THF feed. After centrifugation, the cells were collected and resuspended in 0.4 mol / L HEPES buffer (pH 7.5) to prepare a cell density OD 600 The concentration of 40 was 40. After high pressure homogenization, the cell lysate was obtained. 30 mL was taken and poured into a round-bottom flask. It was incubated in a 37°C constant temperature water bath for 10 min. Then 10 g / L THF, 100 mmol / L MSDS and 1.0 g / L DTT were added successively. The magnetic stirring was immediately turned on to mix evenly. The reaction start was recorded and timed. After 5 h of catalytic reaction, the L-5-MTHF yield was 9.9 g / L and the conversion rate reached 99%.

[0062] The catalytic reaction was carried out with 15 g / L THF feed. After centrifugation, the cells were collected and resuspended in 0.4 mol / L HEPES buffer (pH 7.5) to prepare a cell density OD 600 The cell lysate was 40, and 30 mL was taken and poured into a round-bottom flask. It was incubated in a 37°C constant temperature water bath for 10 min, and then 15 g / L THF, 150 mmol / L MSDS and 1.0 g / L DTT were added. Immediately turn on the magnetic stirring to mix evenly, record the start of the reaction and time it. After 10 h of catalytic reaction, the yield of L-5-MTHF was 14.9 g / L, and the conversion rate reached 99.4%.

[0063] The catalytic reaction was carried out with 20 g / L THF feed. After centrifugation, the cells were collected and resuspended in 0.4 mol / L HEPES buffer (pH 7.5) to prepare a cell density OD 600The cell lysate was 40, and 30 mL was taken and poured into a round-bottom flask. It was incubated in a 37°C constant temperature water bath for 10 min, and then 20 g / L THF, 150 mmol / L MSDS and 1.0 g / L DTT were added successively. Immediately turn on the magnetic stirring to mix evenly, record the start of the reaction and time it. After 12 hours of catalytic reaction, the yield of L-5-MTHF was 17.1 g / L, and the conversion rate reached 85%.

[0064] The catalytic reaction was carried out with 30 g / L THF feed. After centrifugation, the cells were collected and resuspended in 0.4 mol / L HEPES buffer (pH 7.5) to prepare a cell density OD 600 The cell lysate was 40, and 30 mL was taken and poured into a round-bottom flask after high-pressure homogenization. It was incubated in a 37°C constant temperature water bath for 10 min, and then 30 g / L THF, 300 mmol / L MSDS and 1.0 g / L DTT were added successively. Immediately turn on the magnetic stirring to mix evenly, record the start of the reaction and time it. The conversion rates of the catalytic reaction 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 one of SEQ ID NOs. 3 to 9.

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

3. The coding gene according to claim 2, characterized in that: The nucleotide sequence of the coding gene is shown in one of SEQ ID NOs. 10 to 16.

4. A recombinant vector containing the coding gene according to claim 3.

5. A genetically engineered bacterium containing the coding gene according to claim 3.

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

7. The use according to claim 6, characterized in that: The application constructs a genetically engineered bacterium containing the mutant encoding gene, and uses wet bacteria obtained by fermentation culture of the genetically engineered bacteria or enzyme-containing cells obtained by crushing the bacteria as a catalyst to methylate THF to obtain L-5-MTHF.

8. The use according to claim 7, characterized in that: The recombinant bacteria E. coli BL21 were constructed using pET28a plasmid and E. coli BL21 host cells.

9. The use according to claim 7 or 8, characterized in that: The catalysis is carried out under the condition of adding dimethylsulfonium chloride as a methyl donor.

10. The use according to 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° C.-50° C., and the product L-5-MTHF can be obtained.

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