Method for obtaining adenosylmethionine synthetase by prokaryotic overexpression and use thereof

Abstract

The present application relates to biotechnology, and particularly to a method for obtaining adenosylmethionine synthetase by prokaryotic overexpression and a method for synthesizing S-adenosylmethionine (SAM) by using the same. The method for obtaining adenosylmethionine synthetase by prokaryotic overexpression uses Escherichia coli as a genetic engineering host bacterium, and then uses the adenosylmethionine synthetase (MAT) gene derived therefrom to obtain an engineering strain by prokaryotic overexpression, so that adenosylmethionine synthetase is obtained by fermentation of the strain, and high-solubility adenosylmethionine synthetase is obtained by DEAE column purification. The present application uses commonly used Escherichia coli as a genetic engineering host bacterium, and then uses the MAT gene derived therefrom to be modified and expressed by genetic engineering means, and is separated and purified by a DEAE column. By adjusting the experimental conditions, the MAT can be combined with the column, and then the excess impurities in the sample are removed, the MAT is separated and purified, and the production efficiency of SAM is improved.

Description

Technical Field

[0001] This invention relates to biotechnology, specifically to a method for obtaining S-adenosylmethionine synthase through prokaryotic overexpression and a method for synthesizing S-adenosylmethionine (SAM) using it. Background Technology

[0002] S-Adenosylmethionine (SAM) has a wide range of applications; therefore, increasing SAM production is crucial for meeting market demand. The main methods for synthesizing SAM include biosynthesis (microbial fermentation, enzyme catalysis) and chemical synthesis. Chemical synthesis, due to its high cost and low yield, has been gradually phased out. While enzyme-catalyzed synthesis yields high purity, its reliance on ATP increases costs. Microbial fermentation, on the other hand, is green, efficient, low-cost, and suitable for large-scale production. Microbial fermentation can be used to express SAT by transferring it into prokaryotic or eukaryotic systems after genetic modification. Currently, both domestically and internationally, S-adenosylmethionine synthase (MAT) is usually transferred into eukaryotic systems for expression. Most methods to increase MAT expression involve fermentation using yeast as the expression system. However, the proteins obtained by this method are mostly inclusion bodies with low soluble expression levels, which is not conducive to protein purification and is cumbersome. There are precedents for constructing recombinant plasmids to synthesize MAT using prokaryotic expression systems, but the subsequent purification steps are complicated, which is not conducive to obtaining MAT and also makes it inconvenient to use MAT as a raw material for large-scale production of SAM. Summary of the Invention

[0003] The purpose of this invention is to provide a method for obtaining S-adenosylmethionine synthase through prokaryotic overexpression and a method for synthesizing S-adenosylmethionine (SAM) using the synthase.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] A method for obtaining S-adenosylmethionine synthase by prokaryotic overexpression involves using Escherichia coli as the genetically engineered host bacterium, then using the S-adenosylmethionine synthase (MAT) gene derived from Escherichia coli for prokaryotic overexpression to obtain an engineered strain. The strain is fermented to obtain S-adenosylmethionine synthase, and highly soluble S-adenosylmethionine synthase is obtained by purification using a DEAE column.

[0006] Furthermore, the MAT fragment in *E. coli* was amplified by PCR and recombinantly expressed in the plasmid pET-28a-MAT. This plasmid was then transformed into *E. coli* for overexpression to obtain an engineered strain. Fermentation of this strain yielded S-adenosylmethionine synthase, which was then purified using a DEAE column to obtain highly soluble S-adenosylmethionine synthase. The amplification primers were:

[0007] Upstream primer: CGCCATATG GCAAAACACCTTTT (The underlined part is the Nde I restriction site);

[0008] Downstream primer: CCC AAGCTT GGGTTACTTCAGACC (The underlined part is the HindIII restriction site)

[0009] The recombinant expression plasmid pET-28a-MAT was introduced into BL21(DE3) Escherichia coli and cultured at 37°C and 220 rpm for 12-14 hours in the presence of LB liquid medium to obtain a bacterial culture containing the recombinant plasmid. The obtained bacterial culture was mixed with Kana resistant medium and liquid medium, and then inoculated into LB liquid medium at a volume ratio of 1-10% and cultured at 37°C with shaking for 4 hours. IPTG (0.2 g / mL) was added and the culture was induced at 30°C with shaking for 4 hours. After culture, the highly soluble S-adenosylmethionine synthase was obtained by purification.

[0010] The LB liquid medium contains Kana resistance, wherein the volume ratio of Kana resistance to LB liquid medium is 1:1000.

[0011] When the bacterial culture is mixed with Kana resistance and liquid culture medium, the Kana: bacterial culture: LB liquid culture medium is mixed in a volume ratio of 1:10:1000.

[0012] The culture medium was added to a buffer solution, centrifuged to collect the supernatant, and the pH was adjusted to 9.0 and the conductivity to 9.39 ms / cm. Then, it was purified by DEAE column ion exchange chromatography, and gradient elution was performed with 0.02M PBS containing 0-1M NaCl at a flow rate of 0.6 mL / min to obtain high-purity, highly soluble S-adenosylmethionine synthase.

[0013] An application of the method described herein, wherein the method obtains the application of S-adenosylmethionine synthase in the synthesis of S-adenosylmethionine (SAM).

[0014] A method for synthesizing S-adenosylmethionine, which utilizes a obtained S-adenosylmethionine synthase to obtain S-adenosylmethionine (SAM) under enzymatic reaction.

[0015] Furthermore, the obtained S-adenosylmethionine synthase is added to the enzymatic reaction system and the enzymatic reaction is carried out at room temperature (25-30℃) for 0.5-1.5 h, and then purified to obtain S-adenosylmethionine (SAM); wherein, the volume ratio of the bacterial supernatant to the enzymatic reaction system for obtaining S-adenosylmethionine synthase by the method of claim 1 is 0.5-1:2.

[0016] The enzymatic reaction was carried out at 30°C for 1 hour, and then the reaction was terminated by adding an equal volume of 20% HClO4 solution.

[0017] The enzymatic reaction system contains 0.1 mol / L Tris, 0.02 mol / L MgCl, 0.1 mol / L KCl, 0.01 mol / L methionine, 0.01 mol / L ATP, 2 μL β-mercaptoethanol, and hydrochloric acid adjusted to pH 7.6.

[0018] The enzymatic reaction solution was purified and eluted using a C18 reversed-phase column. The purification and elution conditions were as follows: flow rate: 1 mL / min, wavelength: 254 nm, column temperature: 30 °C, mobile phase: 0.01 M ammonium formate, pH adjusted to 3.0 with formic acid, containing a final concentration of 15% ethanol. The peak at approximately 3.4 minutes of HPLC was collected, yielding S-adenosylmethionine (SAM).

[0019] Advantages of this invention:

[0020] This invention uses Escherichia coli, a commonly used genetic engineering host bacterium, and utilizes the MAT gene derived from it to modify and express it through genetic engineering. The MAT gene is then separated and purified using a DEAE column. By adjusting experimental conditions to allow MAT to bind to the column, excess impurities in the sample are removed, thus improving the production efficiency of SAM. This method is suitable for both laboratory research and industrial production, and it is simple and convenient. Attached Figure Description

[0021] Figure 1 This is an electrophoresis diagram of PCR amplification of the MAT gene provided in an embodiment of the present invention.

[0022] Figure 2 Electrophoresis diagram of plasmid construction for double enzyme digestion verification provided in this embodiment of the invention.

[0023] Figure 3 Electrophoresis diagram of MAT soluble analysis provided in the embodiments of the present invention; M marker; Lane 1: Uninduced recombinant bacteria; Lane 2: Induced recombinant bacteria; Lane 3: Induced recombinant bacteria supernatant; Lane 4: Induced recombinant bacteria precipitate.

[0024] Figure 4 The HPLC chromatogram of 0.04 g of S-adenosylmethionine, a standard provided in this embodiment of the invention, dissolved in 2 mL of water.

[0025] Figure 5 The HPLC chromatogram of the supernatant of the 2 mL reaction system of the control group provided in this embodiment of the invention is shown.

[0026] Figure 6The HPLC chromatogram of 1 mL of supernatant after adding 2 mL of bacterial cell lysis to the 2 mL reaction system provided in this embodiment of the invention.

[0027] Figure 7 The HPLC chromatogram of 0.5 mL of the supernatant after crushing was added to the 2 mL reaction system provided in this embodiment of the invention.

[0028] Figure 8 The HPLC chromatogram of 0.01g ATP dissolved in 2mL water is provided for an embodiment of the present invention.

[0029] Figure 9 The HPLC chromatogram of 0.003g methionine dissolved in 2mL water is provided for an embodiment of the present invention.

[0030] Figure 10 The separation and purification of MAT provided in this embodiment of the invention are analyzed by SDS-PAGE electrophoresis; M: marker; 1: original sample; 2: permeate; 3: 0.02M PBS 6.6; 4: 0.02M PBS 6.6 containing 0.3M NaCl; 5: 0.02M PBS 6.6 containing 0.6M NaCl; 6: 0.02M PBS 6.6 containing 1M NaCl.

[0031] Figure 11 SDS-PAGE electrophoresis analysis of nickel column purified MAT provided in this embodiment of the invention; M: marker; 1: original sample; 2: permeate; 3: 0.02M PBS 8.0+0.5; 4: 0.02M PBS 8.0+0.5 containing 4% Triton; 5: 25mM imidazole (PBS 8.0+0.5); 6: 0.02M PBS 6.0+0.5mol NaCl; 7: 0.02M PBS 5.0+0.5mol NaCl; 8: 0.1M sodium citrate buffer pH 3.0+1mol NaCl.

[0032] Figure 12 The diagram shows a comparison of the column loading effects of two purification methods provided in this embodiment of the invention; lane 1 (sample lane) and lane 2 (permeation lane) are for samples purified by DEAE column; lanes 3 and 4 are for samples purified by nickel column and permeation lane, respectively. Detailed Implementation

[0033] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0034] The strains and plasmids used in the following examples, namely pET-28a empty plasmid, BL21(DE3), and DH5α, are all commercially available and are stored in the applicant's laboratory for future use.

[0035] All reagents used in the PCR reaction and T4 DNA ligase were manufactured by Takara Corporation; DNA gel extraction kits and plasmid mini-preparation kits were manufactured by Kangwei Reagent Biotechnology Co., Ltd.; and all other products were domestically produced analytical grade.

[0036] Example 1

[0037] 1) Primer design and synthesis

[0038] The gene for S-adenosylmethionine synthase (MTA) from E. coli was retrieved through NCBI (GenBank ID: 242378468), primers were designed, and the synthesis was commissioned to Beijing Liuhe BGI Genomics Co., Ltd.

[0039] Upstream primer: CGC CATATG GCAAAACACCTTTT (The underlined part is the Nde I restriction site);

[0040] Downstream primer: CCC AAGCTT GGGTTACTTCAGACC (The underlined part is the HindIII restriction site)

[0041] 2) Amplification of the target fragment of MAT

[0042] Weigh 0.12g Tris and 0.03g EDTA and dissolve them in 100mL distilled water. Adjust the pH to 8.0 with HCl to prepare TE buffer. Add 30μL of BL21(DE3) bacterial culture to 3mL LB medium and incubate overnight. Transfer 200μL of the overnight cultured BL21(DE3) to a 1.5mL centrifuge tube and centrifuge at 3500rpm for 5min. Discard the supernatant, add 200μL of TE buffer, mix well, and centrifuge at 3500rpm for 5min. Repeat this step twice. Discard the supernatant, add 200μL of TE buffer, and mix well. Boil in a water bath for 5min, then freeze at -20℃ for 5min. Repeat this step once. Centrifuge at 5000rpm for 1min, collect the supernatant, and obtain the BL21(DE3) DNA solution for later use. Using the BL21(DE3) DNA solution as a template, perform PCR amplification of the target gene fragment using the primers constructed above. The reaction system is shown in Table 1-1, and the program settings are shown in Table 1-2. PCR results were verified using agarose gel electrophoresis on a 0.7% gel. DNA fragments were recovered according to the DNA gel recovery kit instructions (see [link to kit]). Figure 1 ).

[0043] Table 1-1 PCR Reaction System

[0044]

[0045] Table 1-2 PCR Program Settings

[0046]

[0047]

[0048] Depend on Figure 1 As can be seen, four PCR reactions were performed, and a clear amplification band was visible at 1152 bp in each reaction, which was consistent with the expected fragment size, proving that the amplification was successful.

[0049] 3) Construction of recombinant plasmids

[0050] The target fragment was double-digested with the empty pET-28a plasmid. The digestion system is shown in Tables 1-3 and 1-4. After digestion at 37℃ for 1 h, agarose gel electrophoresis was performed and the fragment was recovered. A 0.5 mL EP tube was taken, and 2 μL of the double-digested pET-28a fragment, 15 μL of the double-digested MAT fragment, 2 μL of T4 Buffe, and 1 μL of T4 ligase were added. After mixing, the cells were ligated at 22℃ for 4 h. 100 μL of DH5α competent cells were thawed in an ice box for 15 min. In a clean bench, the enzyme-linked product was added to the EP tube containing 100 μL of DH5α competent cells, gently mixed, and incubated on ice for 30 min, followed by a 90 s water bath at 42℃, and then an ice bath for 2 min. 1 mL of liquid culture medium was added to the EP tube, and the cells were incubated at 37℃ for 1 hour, followed by centrifugation at 3500 rpm for 5 min. Discard the supernatant in a clean bench, homogenize the bacterial cells with a pipette, and transfer them to a culture dish containing Kana resistance. Spread the mixture evenly with a rake. Incubate at 37°C, first upright for 10 min, then inverted for 12 h. Pick two single colonies and add them to two separate 3 mL LB broth media containing 3 μL of 50 μg / mL Kana. Incubate at 37°C and 220 rpm for 12 h. Extract plasmids using a plasmid extraction kit, and verify successful construction by agarose gel electrophoresis after NdeI and HindIII digestion (see [link to relevant documentation]). Figure 2 The constructed recombinant expression plasmid pET-28a-MAT was double-digested and analyzed by 0.7% agarose gel electrophoresis. Large fragments and the target fragment at 1152 bp were observed, confirming that the MAT target fragment was correctly inserted into the vector. After successful verification, it was sent to Beijing BGI Genomics Co., Ltd.

[0051] Table 1-3 pET-28a plasmid digestion system

[0052]

[0053] Table 1-4 MAT enzyme digestion system

[0054]

[0055] 4) Solubility analysis

[0056] The correctly sequenced plasmid was transformed into competent E. coli BL21(DE3). Two single colonies were picked and added to two separate 3 mL LB liquid media containing 3 μL of 50 μg / mL Kana. The cultures were incubated at 37°C and 220 rpm for 14 hours to obtain the recombinant plasmid culture. The recombinant plasmid culture was then inoculated at 1% (v / v) into two new 4 mL LB liquid media containing 0.1% (v / v) Kana antibiotic, resulting in an induction group (2) and a non-induction group (1). The cultures were incubated at 37°C with shaking at 220 rpm. To reduce inclusion body formation, bacterial growth was carried out at 37°C for the first 4 hours, and product synthesis was carried out at 30°C for the next 4 hours. When the bacterial concentration OD... 600 When the concentration reached 0.6-0.8, IPTG with a final concentration of 200 mM was added to the induction group, and the induction culture was continued at 30℃ for 4 h to obtain the induced recombinant bacterial solution. The non-inducible group was the same as the induction group except that no IPTG was added. After the culture was completed, the bacterial cells of the non-inducible group and the inducible group were collected by centrifugation at 3500 rpm for 20 min (4), the supernatant was discarded, and 1 mL of filtered water was added to suspend the bacterial cells. 20 μL of each tube was taken for sample preparation. The remaining suspended bacterial cells of the induction group were broken by sonication and centrifuged at 10000 rpm for 5 min. The supernatant was retained and 20 μL was taken for sample preparation (3). The precipitate was resuspended in 1 mL of filtered water and then 20 μL was taken for sample preparation. After sample preparation, the four samples were detected by 12.5% ​​SDS-PAGE gel electrophoresis to determine the protein expression form (see Figure 3 ).

[0057] Depend on Figure 3 As can be seen, the MAT produced after induction of recombinant bacteria was in the supernatant. Analysis using Imagej software showed that 72% of the protein was soluble. The production and purification of MAT can be verified by fermentation, lysis, and taking the supernatant.

[0058] 5) Fermentation and crushing of microbial cells

[0059] The Kana:recombinant plasmid culture medium:LB liquid medium were mixed at a ratio of 1:10:1000 (volume ratio (μL)). Then, 1% (volume) was inoculated into 50 mL of LB liquid medium containing Kana and cultured at 37°C for 4 h to the logarithmic growth phase. IPTG was added to a final concentration of 200 mM, and the culture was incubated at 30°C for 4 h to obtain the induced cell fermentation broth. The broth was then centrifuged at 3500 rpm for 20 min at 4°C, repeated three times. The precipitate was dispersed and mixed with 0.9% NaCl to collect the cells. 6) DEAE column ion exchange chromatography purification of MAT

[0060] Add 30 mL of 0.02 M pH 9.0 PBS to the induced bacterial cells obtained by centrifugation in step 5) above for disruption (4 s disruption, 16 s pause, 8 min total disruption), centrifuge (10000 rpm, 4℃, 30 min), dilute the supernatant with filtered water to 200 mL, and adjust the pH to 9.0 and conductivity to 9.39 ms / cm with NaOH. Then wash and equilibrate the DEAE chromatography column (1 cm inner diameter, 10 cm height) with 5 column bed volumes of filtered water and 0.02 M pH 9.0 PBS, setting the flow rate to 1 mL / min. After equilibration of the column, sample was loaded at a flow rate of 0.6 mL / min. After loading, the column was washed with 0.02 M pH 9.0 PBS solution until the baseline stabilized. Elution was then performed sequentially using 0.02 M pH 6.6 PBS, 0.02 M PBS 6.6 containing 0.3 M NaCl, 0.02 M PBS 6.6 containing 0.6 M NaCl, and 0.02 M PBS 6.6 containing 1 M NaCl. After elution, the column was washed with 5 column volumes of filtered water. The column was then sealed with 20% ethanol (see [link to column description]). Figure 10 ).

[0061] MAT protein was subjected to ion exchange chromatography on a DEAE column, and the effluent under various conditions was analyzed by SDS-PAGE. Based on the results... Figure 10 MAT was successfully purified in lane 6, with a molecular weight of 45.9 kDa. ImageJ analysis showed a purity of 75%.

[0062] Comparative Example 1

[0063] Because a 6×His tag was incorporated into the experimental design, the commonly used purification method for this type of protein is metal ion chelation chromatography.

[0064] The nickel column purification protocol and its results are shown below:

[0065] Packing material: Chelating Sepharose Fast Flow

[0066] Chromatography column specifications: inner diameter 1cm, column height 8cm

[0067] Flow rate: 1 mL / min

[0068] Mix Kana: bacterial suspension of recombinant plasmid: LB liquid medium in a volume ratio of 1:10:1000 (μL). Inoculate the mixture at a 1% inoculation rate into 6 mL of LB liquid medium containing Kana and incubate overnight at 37°C and 220 rpm for 12-14 h.

[0069] The culture medium obtained above was transferred to a new 500 mL LB liquid medium containing Kana at a 1% volume inoculation rate. After incubation at 37°C and 220 rpm for 4 h with shaking, 125 μL of IPTG was added at a ratio of 1:4000 (v / v), and the culture was induced at 30°C and 220 rpm for 4 h. After centrifugation of 500 mL of fermentation broth, 35 mL of 0.02 M PBS containing 0.5 M NaCl (pH 8.0) was added to the collected bacterial pellet, and the mixture was sonicated for 4 s, followed by a 16 s pause, for a total of 8 min. After sonication, the mixture was centrifuged at 4°C and 10000 rpm for 30 min, and the supernatant was collected as a sample. The column was then processed and eluted according to the method described in the table below. (See table below for reference.) Figure 11 .

[0070] Follow the steps in the table, washing from top to bottom.

[0071]

[0072] Depend on Figure 11 The samples from each lane are listed in the table below. As can be seen from the figure: based on the first two lanes, the target protein did not adhere significantly to the column, and in subsequent lanes, only a significant amount of other proteins were eluted, with no significant target protein appearing at 45 kDa. Furthermore, by comparing the elution effect of the comparative example with the elution and purification effect of the example, [further details are needed]. Figure 11 As can be seen in lane 1 (sample) and lane 2 (through), MAT either does not adhere to the column or adheres very little. For a comparison of the elution results of the two lanes with the elution results of the example, see [reference needed]. Figure 12 As shown in the figure, lane 1 (sample lane) and lane 2 (permeate lane) are for samples purified by the DEAE column; lanes 3 and 4 are for samples purified by the nickel column and permeate lane, respectively. It is clear that the DEAE column has a better column adhesion effect for purifying MAT. 7) HPLC verification of the S-adenosylmethionine enzymatic reaction

[0073] The reaction system solution contains 0.1 mol / L Tris, 0.02 mol / L MgCl, 0.1 mol / L KCl, 0.01 mol / L methionine, 0.01 mol / L ATP, and 2 μL β-mercaptoethanol. Finally, the pH is adjusted to 7.6 with hydrochloric acid for later use.

[0074] Add 15 mL of filtered water to the bacterial cells collected in step 5), break them up, centrifuge at 12000 rpm for 20 minutes to obtain 15 mL of bacterial supernatant containing enzymes.

[0075] The occurrence of the S-adenosylmethionine enzymatic reaction was verified by preparing the following 6 groups of samples. The enzymatic reaction conditions were as follows: after the enzymatic reaction was carried out at 30℃ for 1 hour, the reaction was terminated by adding an equal volume of 20% HClO4 solution.

[0076] The six sample groups consisted of a standard (0.04 g S-adenosylmethionine dissolved in 2 mL of water), a control group (2 mL reaction system without adding supernatant after bacterial cell disruption), a 2 mL reaction system with 1 mL of the above enzyme-containing bacterial supernatant added, a 2 mL reaction system with 0.5 mL of the above enzyme-containing bacterial supernatant added, 0.01 g ATP dissolved in 2 mL of water, and 0.003 g methionine dissolved in 2 mL of water.

[0077] The reaction solutions of the above experimental groups were passed through a C18 reversed-phase column with a flow rate of 1 mL / min, a wavelength of 254 nm, a column temperature of 30 °C, and a mobile phase of 0.01 M ammonium formate. The pH was adjusted to 3.0 with formic acid, and the mobile phase contained 15% ethanol at a final concentration. (See [link to article]) Figure 4-9 (and Tables 2-1 to 2-6).

[0078] Table 2-1 Spectral Record of 0.04 g S-adenosylmethionine Standard Dissolved in 2 mL of Water

[0079]

[0080] Table 2-2 Spectral records of the supernatant after sterile cell lysis in the 2mL reaction system of the control group.

[0081]

[0082] Table 2-3 Spectral Records of 1 mL of Supernatant after Adding 2 mL of Bacterial Cell Disruption to the Reaction System

[0083]

[0084] Table 2-4 Spectral Records of 2 mL Reaction System After Adding 0.5 mL of Supernatant After Crushing

[0085]

[0086] Table 2-5 Spectral Record of 0.01g ATP Dissolved in 2mL of Water

[0087]

[0088] Table 2-6 Spectral Record of 0.003g Methionine Dissolved in 2mL of Water

[0089]

[0090] the following Figure 4-9The HPLC chromatograms for each experimental group are shown in Tables 2-1 to 2-6. A comprehensive analysis of the experimental groups reveals that the retention time of S-adenosylmethionine is approximately 3.4 min; the retention time of ATP is approximately 2.9 min; and the retention time of methionine is approximately 3.6 min. Therefore, the experimental procedures of this invention successfully synthesize the SAM product and verify the occurrence of the enzymatic reaction catalyzed by MAT in the reaction of L-methionine and ATP to produce S-adenosylmethionine (SAM) and pyrophosphate.

[0091] Furthermore, this invention uses *E. coli* as a commonly used host bacterium for genetic engineering, and its derived MAT gene can be modified and expressed through genetic engineering to improve the production efficiency of SAM. In the above embodiment, the MAT fragment in *E. coli* was amplified by PCR, and the recombinant expression plasmid pET-28a-MAT was successfully constructed. The recombinant plasmid was verified by double enzyme digestion and sequencing, confirming its correct construction. It was then transformed into BL21(DE3). To reduce inclusion body formation, bacterial growth was carried out at 37°C for the first 4 hours, followed by MAT synthesis at 30°C for the next 4 hours after the addition of IPTG. SDS-PAGE analysis showed that the expressed protein was mainly expressed in soluble form, accounting for 72% of the total bacterial protein. The purity of the final MAT obtained after ion exchange chromatography using a DEAE column was >75%. After fermentation, centrifugation, and disruption, HPLC was used to verify the occurrence of the enzymatic reaction, demonstrating that the purified MAT has the activity of catalyzing the conversion of L-methionine to S-adenosylmethionine.

Claims

1. A method for obtaining S-adenosylmethionine synthase via prokaryotic overexpression, characterized in that: Using Escherichia coli as the host bacterium for genetic engineering, the engineered strain was obtained by prokaryotic overexpression of the S-adenosylmethionine synthase (MAT) gene derived from it. The strain was fermented to obtain S-adenosylmethionine synthase, and highly soluble S-adenosylmethionine synthase was obtained by purification through a DEAE column.

2. The method for obtaining S-adenosylmethionine synthase by prokaryotic overexpression according to claim 1, characterized in that: The MAT fragment in *E. coli* was amplified by PCR and recombinantly expressed in the plasmid pET-28a-MAT. This plasmid was then transformed into *E. coli* for overexpression to obtain an engineered strain. Fermentation of this strain yielded S-adenosylmethionine synthase, which was then purified using a DEAE column to obtain highly soluble S-adenosylmethionine synthase. The amplification primers were: Upstream primer: CGC CATATG GCAAAACACCTTTT (The underlined part is the Nde I restriction site); Downstream primer: CCC AAGCTT GGGTTACTTCAGACC (the underlined part is the HindIII restriction site).

3. The method for obtaining S-adenosylmethionine synthase by prokaryotic overexpression according to claim 1 or 2, characterized in that: The recombinant expression plasmid pET-28a-MAT was introduced into BL21(DE3) Escherichia coli and cultured at 37°C and 220 rpm for 12-14 hours in the presence of LB liquid medium to obtain a bacterial culture containing the recombinant plasmid. The obtained bacterial culture was mixed with Kana resistant medium and liquid medium, and then inoculated into LB liquid medium at a volume ratio of 1-10% and cultured at 37°C with shaking for 4 hours. IPTG (0.2 g / mL) was added and the culture was induced at 30°C with shaking for 4 hours. After culture, the highly soluble S-adenosylmethionine synthase was obtained by purification.

4. The method for obtaining S-adenosylmethionine synthase by prokaryotic overexpression according to claim 3, characterized in that: The culture medium was added to a buffer solution, centrifuged to collect the supernatant, and the pH was adjusted to 9.0 and the conductivity to 9.39 ms / cm. Then, it was purified by DEAE column ion exchange chromatography, and gradient elution was performed with 0.02M PBS containing 0-1M NaCl at a flow rate of 0.6 mL / min to obtain high-purity, highly soluble S-adenosylmethionine synthase.

5. An application of the method according to claim 1, characterized in that: The method of claim 1 provides the application of S-adenosylmethionine synthase in the synthesis of S-adenosylmethionine (SAM).

6. A method for synthesizing S-adenosylmethionine, characterized in that: S-Adenosylmethionine (SAM) is obtained by using the adenosylmethionine synthase obtained in claim 1 under enzymatic reaction.

7. The method for synthesizing S-adenosylmethionine according to claim 6, characterized in that: The adenosylmethionine synthase obtained by the method of claim 1 is added to the enzymatic reaction system and the enzymatic reaction is carried out at room temperature (25-30℃) for 0.5-1.5 h, and then purified to obtain S-adenosylmethionine (SAM); wherein, the volume ratio of the bacterial supernatant of the adenosylmethionine synthase obtained by the method of claim 1 to the enzymatic reaction system is 0.5-1:

2.

8. The method for synthesizing S-adenosylmethionine according to claim 7, characterized in that: The enzymatic reaction system contains 0.1 mol / L Tris, 0.02 mol / L MgCl, 0.1 mol / L KCl, 0.01 mol / L methionine, 0.01 mol / L ATP, 2 μL β-mercaptoethanol, and hydrochloric acid adjusted to pH 7.

6.

9. The method for synthesizing S-adenosylmethionine according to claim 7, characterized in that: The enzymatic reaction solution was purified and eluted using a C18 reversed-phase column. The purification and elution conditions were as follows: flow rate: 1 mL / min, wavelength: 254 nm, column temperature: 30 °C, mobile phase: 0.01 M ammonium formate, pH adjusted to 3.0 with formic acid, containing a final concentration of 15% ethanol; the peak at approximately 3.4 minutes of HPLC was collected to obtain S-adenosylmethionine (SAM).

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