An O-methyltransferase mutant, its preparation method and application

Abstract

The present invention relates to an O-methyltransferase mutant, and the amino acid sequence of the transaminase mutant is an amino acid sequence mutated from the amino acid sequence shown in SEQ ID NO: 6; wherein, the mutated amino acid sequence has at least one of the following mutation sites: positions 30, 54, 56, 109, 121, 162, 164, 187, 188, 207, 214, 246, 249, 250, 253, 255, 389; the amino acid sequences of the O-methyltransferase mutants are as shown in SEQ ID NOs: 12 to 30, wherein the mutant with the amino acid sequence shown in SEQ ID NO: 30 has high thermal stability and has high catalytic activity for the reaction of producing 6S-5-methyltetrahydrofolic acid with p-methoxybenzoic acid as the substrate, and the product concentration can reach more than 40 g / L, far higher than the results of producing 6S-5-methyltetrahydrofolic acid by synthetic biology reported at home and abroad.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotransformation, and particularly relates to an O-methyltransferase mutant for catalytic preparation of 6S-5-methyltetrahydrofolic acid, a preparation method thereof, and applications thereof. Background Art

[0002] O-methyltransferases are a class of enzymes that catalyze methyl transfer reactions and replace the hydroxyl group of the substrate with a methoxy group. These enzymes play various roles in living organisms. The range of substrates that O-methyltransferases can act on is very wide, including hormone synthesis modification, protein modification, and the synthesis of plant flavonoids. For example, catechol-O-methyltransferase exists in the human body and plays a role in metabolizing catechol neurotransmitters; its function is to methylate the hydroxyl group at the C-3 position of the benzene ring of catecholamines with S-adenosylmethionine as the coenzyme providing the active methyl group under the assistance of magnesium ions. Dopamine modified by O-methylation will be further inactivated in the body; since dopamine is related to mental activities and body movements, catechol-O-methyltransferase is of research significance for neurological diseases and movement disorder diseases such as Parkinson's disease and schizophrenia.

[0003] Folic acid (vitamin B9) is a water-soluble vitamin, which is a collective term for pteroylmonoglutamic acid and compounds related to the biological activity of folic acid. Active folic acid, that is, folic acid with physiological activity itself, can be directly absorbed and utilized, referring to a group of substances, including dihydrofolic acid, tetrahydrofolic acid, 5,10-methylenetetrahydrofolic acid, 10-formylfolic acid, and 6S-5-methyltetrahydrofolic acid, etc. 6S-5-methyltetrahydrofolic acid is the most active form of folic acid among them, is an essential basic substance for human life activities, is the key product of the activation metabolism of synthetic folic acid, and is also the main component of natural folic acid. 6S-5-methyltetrahydrofolic acid (L-5-methyltetrahydrofolic acid) is the only active form of folic acid that plays a role in the human body, can prevent neural tube defects in newborns, and has the effect of preventing and treating Alzheimer's disease. However, the human body itself cannot naturally synthesize 6S-5-methyltetrahydrofolic acid and must obtain it from food. Currently, the chemical synthesis of 6S-5-methyltetrahydrofolic acid will cause serious environmental pollution, resulting in challenges in the supply of green and sustainable 6S-5-methyltetrahydrofolic acid. Compared with chemical synthesis, the biosynthesis method is an important way to achieve the green and sustainable supply of 6S-5-methyltetrahydrofolic acid.

[0004] In recent years, researchers have also begun to use biosynthesis methods to prepare 5-methyltetrahydrofolic acid in order to solve problems such as environmental pollution impact, process impurity limitations, and safety issues during the industrial chemical synthesis of 6S-5-methyltetrahydrofolic acid. For example, in patent CN202310199689A, Lactobacillus plantarum inm30-LP that produces 6S-5-methyltetrahydrofolic acid was reported, and this strain can accumulate folic acid and 5-methyltetrahydrofolic acid during the fermentation process. In patent CN202111385389A, a Bacillus amyloliquefaciens strain was reported, which can also synthesize 5-methyltetrahydrofolic acid under certain growth conditions. In patents CN201210245563A, CN202110672088A, and CN201610325584A, methods for synthesizing 5-methyltetrahydrofolic acid by genetically engineered Escherichia coli strains were reported. In patents WO2021036348A1, CN201811547725A, and CN202010096078A, methods for producing 5-methyltetrahydrofolic acid by metabolically engineering Bacillus subtilis were reported. In patent CN201811547736A, a method for expressing 5,10-methylenetetrahydrofolate reductase and serine hydroxymethyltransferase genes in Lactococcus lactis to accumulate 5-methyltetrahydrofolic acid in its cells was reported. However, a major problem with the above biological methods for producing 6S-5-methyltetrahydrofolic acid is that the yield of the target product is extremely low, only dozens of milligrams per liter, and the highest yield is 306.5 mg / L. This results in high production costs, complex purification processes, and high impurities of 5-methyltetrahydrofolic acid, making industrial production difficult.

[0005] In view of the defects of the above methods for preparing 6S-5-methyltetrahydrofolic acid, the present invention provides an O-methyltransferase mutant and its application in the preparation of 6S-5-methyltetrahydrofolic acid. The method for preparing 5-methyltetrahydrofolic acid using the O-methyltransferase mutant can achieve the preparation of a single configuration of 6S-5-methyltetrahydrofolic acid. This method is safe, environmentally friendly, and low-cost, and can achieve a yield of 42 g / L. Summary of the Invention

[0006] The purpose of the present invention is to provide an O-methyltransferase mutant with high catalytic activity and good thermal stability for the methyl transfer reaction with p-methoxybenzoic acid as the substrate, its preparation method, and its application in the process of preparing 6S-5-methyltetrahydrofolic acid.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The inventors' research found that all the enzymes in Table 1 can catalyze the transfer of the methyl group in vanillic acid (Formula 1) and syringic acid (Formula 2) to tetrahydrofolic acid with different activities, generating 5-methyltetrahydrofolic acid. However, the high prices of vanillic acid and syringic acid lead to high production costs of 5-methyltetrahydrofolic acid. To reduce the production cost of 5-methyltetrahydrofolic acid, the inventors tried to use other methyl donors, including p-methoxybenzoic acid (Formula 3) and m-methoxybenzoic acid (Formula 4). As shown in Table 1, some enzymes have high activities towards m-methoxybenzoic acid, but almost all enzymes have low activities towards the lower-cost p-methoxybenzoic acid. In Table 1, "-" means that when detecting the enzyme activity according to Example 5, no 6S-5-methyltetrahydrofolic acid is generated when the enzyme catalyzes in the reaction system for 4 h; "+" means that when the enzyme catalyzes in the reaction system for 4 h, 0-20% of folic acid is converted into 6S-5-methyltetrahydrofolic acid; "++" means that when the enzyme catalyzes in the reaction system for 4 h, 20-40% of folic acid is converted into 6S-5-methyltetrahydrofolic acid; "+++" means that when the enzyme catalyzes in the reaction system for 4 h, 40-60% of folic acid is converted into 6S-5-methyltetrahydrofolic acid; "++++" means that when the enzyme catalyzes in the reaction system for 4 h, 60-80% of folic acid is converted into 6S-5-methyltetrahydrofolic acid; "+++++" means that when the enzyme catalyzes in the reaction system for 4 h, 80-100% of folic acid is converted into 6S-5-methyltetrahydrofolic acid.

[0009]

[0010] Table 1 Enzyme activities of O-methyltransferases with SEQ ID NOs 1-11 towards different methyl donors

[0011]

[0012] To enable the enzyme to more effectively catalyze the transfer of the methyl group on p-methoxybenzoic acid to tetrahydrofolic acid, the present invention combines rational design, directed evolution, and high-throughput screening techniques of the enzyme to modify the enzyme protein. During the modification process, when the amino acids at positions 30, 54, 56, 109, 121, 162, 164, 187, 188, 207, 214, 246, 249, 250, 253, 255, and 389 of the O-methyltransferase of SEQ ID NO 6 are mutated, it will change its catalytic activity towards the reactions with substrates of vanillic acid, syringic acid, p-methoxybenzoic acid, and m-methoxybenzoic acid, especially increasing the catalytic activity of the enzyme towards the reaction with p-methoxybenzoic acid as the substrate. Preferably, the mutant shown in SEQ ID NO: 30 has high thermal stability and high catalytic activity towards the reaction with p-methoxybenzoic acid as the substrate, while having low activity towards m-methoxybenzoic acid as the substrate, and its product is 6S-5-methyltetrahydrofolic acid.

[0013] According to another aspect of the present invention, there are provided O-methyltransferase mutants shown in SEQ ID NO: 12 to SEQ ID NO: 30, and their thermal stability is also significantly improved relative to their wild O-methyltransferase.

[0014] An O-methyltransferase involved in the present invention is derived from Sphingobium indicum, and its wild-type amino acid sequence is as shown in SEQ ID NO 6. "Wild-type" refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism, which can be isolated from a natural source and has not been deliberately modified by human manipulation. The enzymes obtained after the expression of these genes have low catalytic activity for certain substrates and poor chiral purity of the products obtained by catalysis.

[0015] The present invention provides some O-methyltransferase mutants, which can change the activity of the enzyme for m-methoxybenzoic acid as a substrate, and in particular can improve the activity of the enzyme to catalyze the methylation of p-methoxybenzoic acid to tetrahydrofolic acid. As shown in Table 2, some O-methyltransferase mutants provided by the present invention have a significantly better half-life at 40 °C than the wild-type O-methyltransferase. In Table 2, "-" means that when detecting the enzyme activity according to Example 5, no 6S-5-methyltetrahydrofolic acid is generated when the enzyme catalyzes in the reaction system for 4 h; "+" means that when the enzyme catalyzes in the reaction system for 4 h, 0-20% of folic acid is converted into 6S-5-methyltetrahydrofolic acid; "++" means that when the enzyme catalyzes in the reaction system for 4 h, 20-40% of folic acid is converted into 6S-5-methyltetrahydrofolic acid; "+++" means that when the enzyme catalyzes in the reaction system for 4 h, 40-60% of folic acid is converted into 6S-5-methyltetrahydrofolic acid; "++++" means that when the enzyme catalyzes in the reaction system for 4 h, 60-80% of folic acid is converted into 6S-5-methyltetrahydrofolic acid; "+++++" means that when the enzyme catalyzes in the reaction system for 4 h, 80-100% of folic acid is converted into 6S-5-methyltetrahydrofolic acid.

[0016] Table 2 Enzyme activities of the O-methyltransferase shown in SEQ ID NO 6 and its mutants

[0017]

[0018]

[0019] The present invention performs mutations on it by rational design (on the basis of understanding the spatial structure of the protein, changing individual amino acids in the protein molecule by site-directed mutagenesis or other methods) and methods such as overlap extension PCR, recombinant PCR, megaprimer PCR, and circular plasmid PCR, so as to obtain the target gene of the O-methyltransferase mutant.

[0020] The present invention provides a recombinant plasmid of an O-methyltransferase mutant gene, which can be constructed by ligating the DNA sequence obtained by codon optimization of the O-methyltransferase gene of the present invention for various species to various prokaryotic expression vectors or eukaryotic expression vectors through conventional methods in the art. Such as prokaryotic expression vectors and eukaryotic expression vectors such as pGEX, pMAL, pET series, etc., and more preferably selected from the pET series. The plasmid used in an embodiment of the present invention is pET-24a.

[0021] The present invention provides a genetically engineered bacterium for producing the O-methyltransferase mutant, and the genetically engineered bacterium contains the O-methyltransferase mutant gene of the present invention or the recombinant vector of the present invention. The host cell of the above-mentioned genetically engineered bacterium is preferably Escherichia coli BL21(DE3).

[0022] The present invention provides a method for preparing the O-methyltransferase mutant, including fermenting and culturing the genetically engineered bacterium, and collecting and preparing the recombinant O-methyltransferase.

[0023] The above method includes the step of industrially preparing the recombinant O-methyltransferase under certain production tank fermentation conditions; the preferred production tank fermentation conditions are: DO above 10%, and the air flow rate is 1:0.5 - 2 vvm.

[0024] The O-methyltransferase mutant of the present invention acts as a catalyst in the methyl transfer reaction, and can be used to transfer methyl to tetrahydrofolic acid using m-methoxybenzoic acid and p-methoxybenzoic acid as substrates in formula (a) and formula (b) to obtain 6S-5-methyltetrahydrofolic acid.

[0025]

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] The present invention provides mutants of O-methyltransferase, and these mutants can improve the enzyme activity towards p-methoxybenzoic acid and are used in the synthesis reaction of 6S-5-methyltetrahydrofolic acid in formula (a) and formula (b).

[0028] The enzyme mutants involved in the present invention have the advantage of good thermal stability, and the half-life at 40°C is better than that of the wild enzyme.

[0029] When using the enzyme mutants involved in the present invention to produce 6S-5-methyltetrahydrofolic acid, the concentration of 6S-5-methyltetrahydrofolic acid at the reaction end point can reach more than 40 g / L, which is much higher than the results of producing 6S-5-methyltetrahydrofolic acid by synthetic biology reported at home and abroad.

[0030] Using the O-methylase involved in the present invention to prepare 6S-5-methyltetrahydrofolic acid, in the production process, p-methoxybenzoic acid with a lower price is used, and the cost advantage is obvious. In addition, in the production process, the emissions of three wastes are less, the reaction conversion rate is high, and it has good application prospects. Description of the Drawings

[0031] Figure 1 HPLC chromatogram of folic acid standard product at 0.5 mg / ml;

[0032] Figure 2 HPLC chromatogram of tetrahydrofolic acid standard product at 0.5 mg / ml;

[0033] Figure 3 HPLC chromatogram of 5-methyltetrahydrofolic acid standard product at 0.5 mg / ml;

[0034] Figure 4 HPLC chromatogram of p-methoxybenzoic acid standard product at 4 mM;

[0035] Figure 5 HPLC chromatogram of the sample at the end of the 2L scale-up reaction;

[0036] Figure 6 HPLC detection chromatogram of the product after extraction and purification of the 2L scale-up reaction;

[0037] Figure 7 NMR analysis result of 6S-5-methyltetrahydrofolic acid. Detailed Description of the Invention

[0038] The exemplary embodiments of the present invention will be described in more detail below with reference to the drawings. Although the exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present invention can be more thoroughly understood and the scope of the present invention can be completely conveyed to those skilled in the art. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the drawings and in combination with the embodiments.

[0039] Example 1: Establishment of a genetically engineered bacterium of wild-type O-methyltransferase enzyme

[0040] The codons of the O-methyltransferase wild-type amino acid sequences (SEQ ID NO 1 to SEQ ID NO 11) were optimized, and then the full gene fragment was artificially synthesized by a gene synthesis company. The gene was inserted into the NdeI and BamHI sites of the pET-24a plasmid, and the ligated vector was transferred into Escherichia coli BL21(DE3) to establish an O-methyltransferase genetic engineering bacterium.

[0041] Example 2: Obtaining the O-methyltransferase mutant gene

[0042] The three-dimensional structure of the O-methyltransferase wild-type gene shown in SEQ ID NO 6 has not been revealed yet. However, in this study, the three-dimensional model of the wild-type gene sequence was constructed using SWISS-MODEL, and it was found to be highly homologous to the aryl O-demethylase (PDB: 5TL4) from Sphingomonas paucimobilis. Therefore, referring to the three-dimensional structure of this enzyme for analysis, using p-methoxybenzoic acid and tetrahydrofolic acid as substrates, the binding simulation with the protein was carried out through Docking software, and finally through Pymol analysis, the amino acids that might be related to the binding of the substrate p-methoxybenzoic acid, the binding of tetrahydrofolic acid, and methyl transfer were selected as the mutant amino acids.

[0043] In addition to the above rational design, in this study, the protein engineering of O-methyltransferase was carried out by the method of error-prone PCR random mutagenesis. Generally speaking, error-prone PCR can change the mutation frequency during the amplification of the target gene by adjusting the reaction conditions (such as increasing the magnesium ion concentration, adding manganese ions, changing the concentration of the four dNTPs in the system, or using a low-fidelity DNA polymerase, etc.) when amplifying the target gene by DNA polymerase, so as to randomly introduce mutations into the target gene at a certain frequency to obtain random mutants of the protein molecule.

[0044] In this study, a lower-fidelity Taq polymerase was used, and at the same time, Mn 2+ was used to replace the natural cofactor Mg 2+ to increase the probability of error-prone.

[0045] The 50 μL PCR system is as follows:

[0046]

[0047] Sterilized double-distilled water was added to make up to 50 μL.

[0048] Among them: The O-methyltransferase template gene was constructed by PCR amplifying the O-methyltransferase gene according to the method of Example 1 and inserting the gene into the pET-24a plasmid; for primer design, in this study, the upstream and downstream sequences of the target gene in the recombinant plasmid constructed in Example 1 were designed.

[0049] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 2.5 min; denaturation at 94°C for 15 s, annealing at 53°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; continued extension at 72°C for 10 min, and cooled to 4°C.

[0050] The obtained PCR amplification product was ligated to the pET-24a vector and transferred into Escherichia coli BL21(DE3) to establish an O-methyltransferase gene mutation library.

[0051] Using Escherichia coli BL21(DE3) as the host and the pET-24a plasmid as the vector, the extended O-methyltransferase was expressed, and the high-activity mutant strains were screened by the enzyme activity detection method described in Example 5. The mutated high-activity O-methyltransferase gene was identified. The amino acid sequences of the screened high-activity O-methyltransferase mutants are shown in SEQ ID NO 12-30.

[0052] Example 3: Small-scale production of O-methyltransferase mutants in shake flasks

[0053] The Escherichia coli containing the recombinant plasmids constructed in Examples 1 and 2 was inoculated into 50 mL of LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.2) containing kanamycin (50 μg / mL). It was shaken and cultured at 37°C and 210 rpm for 16 hours. Then it was transferred at a ratio of 1:100 into 100 mL of LB medium containing kanamycin, shaken and cultured at 37°C and 210 rpm, and the absorbance value (OD600) of the bacterial solution at 600 nm was measured regularly to monitor the cell growth density. When the OD600 of the culture was 0.6-0.8, isopropyl β-D-thiogalactoside (IPTG) with a final concentration of 0.8 mM was added to induce the expression of the target O-methyltransferase gene, and the induction culture was carried out overnight (≥16 hours). It was centrifuged at 10000 rpm and 4°C for 10 min, the supernatant was discarded, the cell precipitate was resuspended in pre-cooled 50 mM Tris-HCl buffer (pH 7.5) at 200 g / L, sonicated, and then centrifuged at 13000 rpm and 4°C for 30 min, and the supernatant was collected, which was the crude enzyme solution and stored at -20°C.

[0054] Example 4: Fermentation production of O-methyltransferase

[0055] The recombinant Escherichia coli constructed in Examples 1 and 2 (containing the mutant O-methyltransferase gene) was inoculated with a single microbial colony into 120 mL of LB medium (containing 50 μg / mL kanamycin), and cultured overnight (≥5 hours) at 37°C with shaking at 210 rpm. Then, fermentation was carried out in a 15 L fermenter: the seed liquid was inoculated into 6 L of fermentation medium at an inoculation amount of 2%, and the pH of the fermentation broth was maintained at 7.0 - 7.2 by adding ammonia water. The temperature of the fermenter was 37°C, the stirring speed was 300 - 900 rpm, and the dissolved oxygen was controlled at about 30% during the process. The air flow rate was 1:0.5 - 2 vvm. After culturing for 8 hours, IPTG was added (final concentration 0.8 mmol / L), and the temperature of the fermenter was adjusted to 22°C, and fermentation was continued for 12 - 16 hours. During the fermentation process, a feeding solution (glucose 200 g / L, yeast extract 100 g / L, pH 7.2) was added to maintain the growth of the culture. After the fermentation was completed, the culture was centrifuged to harvest the cells, and then the cells were resuspended in 50 mM phosphate buffer at pH 7.5 to 300 g / L (wet cell weight). After thorough stirring and mixing, the cells were homogenized and disrupted with a high-pressure homogenizer. To the disrupted fermentation broth, polyethyleneimine with a final concentration of 2 g / L and diatomaceous earth with a final concentration of 150 g / L were added, and the mixture was stirred for 30 minutes. After the flocculation and sedimentation were completed, filtration was carried out using a filter cloth lined with diatomaceous earth. The filtered enzyme solution was filtered and concentrated with an ultrafiltration membrane to prepare a crude O-methyltransferase enzyme solution, which was stored at -20°C.

[0056] Example 5: Determination of the enzyme activity of O-methyltransferase and its mutant

[0057] The enzyme activity of O-methyltransferase was determined using a microplate reader, and the determination system is shown in Table 3.

[0058] Table 3 Components of the O-methyltransferase activity detection system

[0059]

[0060]

[0061] 250 μL was dispensed into each well, and finally 50 μL of the O-methyltransferase enzyme solution was added using a multi-channel pipette. In the control wells, Tris-Hcl 7.5 with the corresponding volume and pH was added. The reaction was carried out in a shaker at 30°C with shaking at 120 rpm for 4 h, and samples were taken respectively. Dilute with the mobile phase by 10 times. Heat denature at 90°C for 5 min to denature and precipitate the protein. After filtration, the sample injection volume was 5 μL, and 5-methyltetrahydrofolate, tetrahydrofolate, and folic acid were detected by HPLC.

[0062] HPLC was used to detect the product formation in the reaction system, and the method is shown in Table 5.

[0063] Table 5 Analytical method for 5-methyltetrahydrofolate and tetrahydrofolate

[0064] Parameter Method Chromatographic column Kromasil 100 - 5 C18 column 250 * 4.6 mm Mobile phase <![CDATA[25 mM NaH2PO4 aqueous solution with 6% acetonitrile]]> Flow rate 0.75 ml / min Column temperature 40℃ Injection volume 5 μL Elution time 20 min

[0065] Folic acid, tetrahydrofolic acid, 5-methyltetrahydrofolic acid, and p-methoxybenzoic acid were detected using the method shown in Table 5, and the chromatogram is as Figures 1 to 4 shown.

[0066] Example 6: Detection of the Half-life of O-Methyltransferase and Its Mutants

[0067] 12 ml of the crude enzyme solution obtained in Example 3 above was aliquoted into 12 tubes, placed in a water bath at 40 °C for incubation, and one tube was taken out every 2 h and cooled in an ice bath. The enzyme activity was detected according to the method described in Example 5. The time when the residual enzyme activity decreased to about 50% of the original enzyme activity was the half-life of the enzyme at this temperature, and the temperature stability of the O-methyltransferase mutant was determined accordingly.

[0068] Table 2 shows the enzyme activity half-lives of wild-type O-methyltransferase and mutants at different temperatures: among them, the half-life of wild-type O-methyltransferase at 40 °C was less than 2 h; while the O-methyltransferase mutants shown in SEQ ID NO: 14-19, SEQ ID NO: 21, and SEQ ID NO: 23-30 had a significantly increased half-life at 40 °C. The O-methyltransferase mutants shown in SEQ ID NO: 29 and 30 had a half-life exceeding 24 h at 40 °C.

[0069] Example 7: Scale-up Reaction at 2 L Scale and Product Extraction

[0070] The O-methyltransferase and other components obtained from a 15 L fermenter were added to a 5 L bioreactor according to Table 4 below.

[0071] Table 4 Enzyme Reaction System of O-Methyltransferase at 2 L Scale

[0072]

[0073] During the reaction process, the pH was controlled to 7.5 with 1 M NaOH, the temperature was controlled at 30 °C, the stirring speed was set at 100 rpm, and nitrogen was bubbled throughout the reaction with a nitrogen flow rate of 0.5 L / min. Starting from 2 h after the reaction, folic acid, glucose, and p-methoxybenzoic acid were added dropwise to the reaction system and added dropwise within 1 h. Samples were taken every hour and diluted 20 times with the mobile phase. Heat denaturation was carried out at 90 °C for 5 min to denature and precipitate the protein. After filtration, the sample injection volume was 5 μl, and 5-methyltetrahydrofolic acid, tetrahydrofolic acid, and folic acid were detected by HPLC.

[0074] The reaction ended after 9 h. It was detected that all the folic acid was consumed and converted into 5-methyltetrahydrofolic acid. 50% sulfuric acid was added to adjust the pH to 2.5 to terminate the reaction. After stirring for 30 min, 1 M NaOH was used to adjust the pH to 7.5 (maintaining the pH unchanged for 15 min).

[0075] The reaction solution was taken out and 40 g / L of diatomaceous earth was added, and the precipitate was removed by filtration. The pH of the supernatant was adjusted to 3.0, and the precipitate was precipitated overnight at 2-8 °C. After filtration, the filter cake was washed twice with absolute ethanol and placed in a vacuum drying oven for vacuum drying at 35 °C for 4 h to obtain 84 g of pure 6S-5-methyltetrahydrofolic acid. The NMR spectrum of the obtained product is as shown in Figure 7 shown.

[0076] When the reaction system in this example was detected by the method shown in Table 4, the chromatogram was as shown in Figure 5 , Figure 6 shown.

[0077] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, various modifications and variations can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An O-methyltransferase mutant, characterized in that, The amino acid sequence of the O-methyltransferase mutant is a mutant obtained by mutating the amino acid sequence shown in SEQ ID NO: 6, and the amino acid sequences of the O-methyltransferase mutants are shown in SEQ ID NO: 21, 29, and 30.

2. An O-methyltransferase mutant gene, characterized in that, The O-methyltransferase mutant gene is used to encode the transferase mutant described in claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains the O-methyltransferase mutant gene described in claim 2.

4. The recombinant expression vector according to claim 3, wherein The recombinant expression vector uses pET-24a as the vector plasmid.

5. A genetically engineered bacterium for producing the O-methyltransferase mutant according to claim 1, characterized in that The genetically engineered bacterium contains the recombinant expression vector described in claim 3, and the host cell of the genetically engineered bacterium is Escherichia coli.

6. Use of the O-methyltransferase mutant gene described in claim 2, the recombinant expression vector described in claim 3, and the genetically engineered bacterium described in claim 5 in the preparation of the O-methyltransferase mutant described in claim 1.

7. A method for preparing the O-methyltransferase mutant according to claim 1, characterized in that, It includes the following steps: culturing the genetically engineered bacterium described in claim 5 to obtain the O-methyltransferase mutant.

8. The preparation method according to claim 7, wherein It includes the step of fermenting and preparing the O-methyltransferase mutant.

9. Use of the O-methyltransferase mutant according to claim 1 in a methylation reaction, characterized in that, The O-methyltransferase mutant is used as a catalyst in the following methyl transfer reaction, The reaction product is 6S-5-methyltetrahydrofolic acid.

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