S-adenosine-L-methionine hydroxide transferase and application thereof

Abstract

The invention discloses an S-adenosine-L-methionine hydroxide transferase and an application of the S-adenosine-L-methionine hydroxide transferase. The amino acid sequence of the S-adenosine-L-methionine hydroxide transferase is as shown in SEQ ID NO. 2. The S-adenosine-L-methionine hydroxide transferase obtained by screening has high heat resistance, and can still keep 50% of activity at a high temperature of 80 DEG C. Meanwhile, the activity of the enzyme is still about 78% after the enzyme is placed at normal temperature for four days, and the enzyme has good stability and maintains good activity under the alkaline condition (pH = 7.0-10.0).

Description

(1) Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to an S-adenosyl-L-methionine hydroxide transferase and its application. (2) Background Art

[0002] S-adenosyl-L-methionine hydroxide adenosyltransferase (HATase), also known as S-adenosylmethionine hydroxylase (SAM hydroxylase), catalyzes the nucleophilic attack of water (hydroxide) at the C5' of SAM to generate adenosine and L-methionine.

[0003] S-adenosyl-L-methionine hydroxide adenosyltransferase was initially derived from the archaeon Pyrococcus-horikoshii-OT3 with an optimal growth temperature of 98°C. It and S-adenosyl-L-methionine-dependent fluorinase / chlorinase both belong to a large microbial superfamily covering more than 100 archaea and bacteria. This superfamily is classified as having an unknown functional protein domain DUF-62 (Pfam 01887, Domain of Unknown Function 62). During each conformational change of S-adenosyl-L-methionine hydroxide transferase, a proton is released. In addition, the enzyme loses its activity at a pH of about 5 and exhibits the highest activity at a pH of 8.5. So far, the genes of DUF-62 have been found in up to 200 microorganisms, most of which are extremophiles. Therefore, S-adenosyl-L-methionine hydroxide transferase may play an important role in regulating the intracellular pH of extremophiles. In terms of the research on extreme environment adaptability, its high-temperature stability provides clues for studying the environmental adaptability of enzymes and guides the optimization of the thermal stability of other industrial enzymes.

[0004] In terms of epigenetic research, S-adenosyl-L-methionine, as a methyl donor, plays a key role in DNA and histone methylation, affecting gene expression regulation. And S-adenosyl-L-methionine hydroxide transferase can indirectly affect epigenetic modifications by regulating the level of S-adenosyl-L-methionine, providing potential targets for the research on epigenetic-related diseases such as cancer and neurodegenerative diseases. In environmental technology, using the nitrogen heterocyclic structure of its product adenosine, it can adsorb heavy metal ions such as Cu 2+ 、Cd 2+ etc.

[0005] Many SAM-dependent enzymes are prone to inactivation in in vitro applications, especially under high-temperature or long-term storage conditions, which limits their practicality in industrial catalysis or drug development. So far, research on SAM hydroxide transferases remains relatively limited. (III) Summary of the Invention

[0006] The object of the present invention is to provide an S-adenosyl-L-methionine hydroxide transferase and its application. This enzyme has advantages such as high temperature resistance and good stability, solving the stability problem in industrial applications. Under industrial conditions such as high temperature, extreme pH, or organic solvents, traditional enzymes are easily inactivated, while enzymes with high temperature resistance and good stability can improve catalytic efficiency and reduce production costs. In biotechnological fields that require high-temperature treatment (such as biofuel production, wastewater treatment), high-temperature-resistant enzymes can significantly improve process efficiency.

[0007] The technical solution adopted by the present invention is as follows:

[0008] In the first aspect, the present invention provides an S-adenosyl-L-methionine hydroxide transferase, and the amino acid sequence of the S-adenosyl-L-methionine hydroxide transferase is as shown in SEQ ID NO.2.

[0009] In the second aspect, the present invention provides a coding gene for the above-mentioned S-adenosyl-L-methionine hydroxide transferase, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.1.

[0010] In the third aspect, the present invention provides a recombinant expression vector containing the coding gene for the S-adenosyl-L-methionine hydroxide transferase. The recombinant expression vector is based on the plasmid pET-28a(+).

[0011] In the fourth aspect, the present invention provides a recombinant genetically engineered bacterium transformed with the above-mentioned recombinant expression vector. The recombinant genetically engineered bacterium uses E.Coli BL21(DE3) as the host bacterium.

[0012] In the fifth aspect, the present invention provides an application of the above-mentioned S-adenosyl-L-methionine hydroxide transferase in catalyzing the preparation of adenosine from S-adenosylmethionine.

[0013] Furthermore, the application is as follows: Using the pure enzyme solution extracted by ultrasonic disruption of the wet cells obtained by inducing and culturing the recombinant gene bacteria containing the coding gene for S-adenosyl-L-methionine hydroxide transferase as a catalyst, using S-adenosylmethionine as a substrate, and using a buffer solution with a pH of 6-10 as a reaction medium to form a reaction system. After the reaction is completed at 20-60°C, the reaction solution is separated and purified to obtain adenosine.

[0014] Further, the final concentration of the substrate added to the reaction system is 300 - 900 μM (preferably 600 μM), and the final concentration of the catalyst added, calculated based on the protein content, is 0.1 - 5 mg / mL (preferably 0.5 mg / mL).

[0015] Further, the reaction system also contains EDTA or metal ions, and the metal ions include Na + 、K + 、Mg 2+ 、Zn 2+ 、Mn 2+ 、Cu 2+ 、Co 2+ 、Ca 2+ ; the final concentration of the added EDTA or metal ions is 0.1 - 0.5 mM (preferably 0.5 mM).

[0016] Further, the reaction is carried out at 25 °C for 1 h.

[0017] Further, the catalyst is prepared as follows:

[0018] (1) Preparation of wet cells: Streak the glycerol stock solution of the recombinant genetic engineering bacterium containing the gene encoding S-adenosyl-L-methionine hydroxide transferase on an LB solid medium plate containing 30 μg / mL kanamycin resistance, and incubate it upside down at 37 °C overnight; pick a single colony and inoculate it into a test tube containing an LB liquid medium with 30 μg / mL kanamycin resistance, and shake it at 37 °C and 200 rpm overnight to obtain the activated seed solution; inoculate the activated seed solution into a 100 mL LB liquid medium shake flask containing 30 μg / mL kanamycin resistance at an inoculation amount of 2% (v / v), shake it at 37 °C and 200 rpm for about 2 h until OD 600 = 0.6 - 0.8, add IPTG with a final concentration of 0.1 mM, and induce it at 18 °C and 120 rpm for 18 - 24 h; centrifuge the induced bacterial solution at 4 °C and 3500 rpm for 20 min using a vertical large-capacity centrifuge, discard the supernatant, and collect the wet cells;

[0019] (2) Preparation of crude enzyme solution: Resuspend the wet cells in PBS, perform ultrasonic disruption on the cell suspension, disrupt it at a power of 40 W for 15 min until the solution becomes clear, with a disruption interval of 3 s and a pause of 5 s, gently mix the sample with a shaker, and then centrifuge the sample at 4 °C and 12000 rpm for 30 min, collect the supernatant and the precipitate, and the supernatant is the crude enzyme solution;

[0020] (3) Preparation of pure enzyme solution: Load Ni-NTA affinity resin into a chromatography column, wash it three times with pure water, eluting 3 column volumes each time; then, perform two equilibration treatments with NTA-0 eluent, 3 column volumes each time, and let it stand after each equilibration, which is the pretreated Ni-NTA resin; Composition of NTA-0 eluent: 20 mM Tris, 500 mM NaCl, 10% (v / v) glycerol, and the solvent is water;

[0021] Mix the above-mentioned crude enzyme solution with the pretreated Ni-NTA resin at a volume ratio of 10:1, and chelate at 4°C for 3 h using an oscillating thermostatic metal bath device; Inject the chelated mixture into the chromatography column and collect the effluent, which is the protein flow-through solution; Subsequently, sequentially elute with NTA-20 eluent, NTA-50 eluent, NTA-100 eluent, and NTA-300 eluent according to a preset gradient, eluting 2 / 3, 1 / 3, 1, and 1 column volume respectively; Collect the effluents of each concentration gradient, verify by SDS-PAGE detection and merge the effluents containing the target protein, and obtain the pure enzyme solution of S-adenosyl-L-methionine hydroxide transferase after ultrafiltration and concentration through a 3 kDa ultrafiltration tube to remove salt;

[0022] NTA-20 eluent: 20 mM Tris, 500 mM NaCl, 10% (v / v) glycerol, 20 mM imidazole, and the solvent is water;

[0023] NTA-50 eluent: 20 mM Tris, 500 mM NaCl, 10% (v / v) glycerol, 50 mM imidazole, and the solvent is water;

[0024] NTA-100 eluent: 20 mM Tris, 500 mM NaCl, 10% (v / v) glycerol, 100 mM imidazole, and the solvent is water;

[0025] NTA-300 eluent: 20 mM Tris, 500 mM NaCl, 10% (v / v) glycerol, 300 mM imidazole, and the solvent is water.

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

[0027] The S-adenosyl-L-methionine hydroxide transferase screened by the present invention has high heat resistance and can still maintain 50% activity at a high temperature of 80°C. At the same time, the activity of this enzyme is still about 78% after being placed at room temperature for four days, has good stability, and maintains good activity under alkaline conditions (pH = 7.0 - 10.0). (IV) Description of the Drawings

[0028] Figure 1, SDS-PAGE electrophoresis diagrams of Protein Flu-3 before and after purification in Example 1; M: Marker; 1: Supernatant of Flu-3 cell disruption; 2: Precipitate of cell disruption; 3: Protein flow-through; 4 / 5: Washing with 20 mM imidazole; 6: Washing with 50 mM imidazole; 7 / 8 / 9: Washing with 100 mM imidazole; 10 / 11 / 12 / 13: Washing with 300 mM imidazole.

[0029] Figure 2 , Reaction schematic diagram of the preparation of adenosine by 5'-substitution catalyzed by S-adenosyl-L-methionine hydroxide transferase Flu-3 using S-adenosyl-L-methionine (SAM) as a substrate.

[0030] Figure 3 , HPLC and LC-MS analysis results of the enzyme reaction in Example 2.

[0031] Figure 4 , Standard curve of adenosine in Example 3.

[0032] Figure 5 , Effect of different pH values on enzyme activity in Example 3.

[0033] Figure 6 , Effect of different temperatures on enzyme activity in Example 3.

[0034] Figure 7 , Effect of different metal ions and chelating agents on enzyme activity in Example 3.

[0035] Figure 8 , Relative activity of the enzyme at different times at 25°C in Example 3. (V) Specific Embodiments

[0036] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0037] Composition of the culture medium used in the embodiments of the present invention:

[0038] Preparation of LB liquid medium: Weigh accurately 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride, add 1 L of deionized water, and stir well to dissolve.

[0039] LB solid medium is prepared by adding 20 g / L of agar to LB liquid medium.

[0040] Example 1: Preparation of S-adenosyl-L-hydroxide transferase

[0041] 1. Construction of recombinant genetic engineering bacteria containing the S-adenosyl-L-methionine hydroxide transferase gene

[0042] Based on the identified S-adenosyl-L-methionine-dependent fluorinase protein sequences, 9,971 potential fluorinases were mined from more than 220,000 bacterial genomes using Hidden Markov Models (HMMs). These candidate fluorinases were mainly annotated as SAM-dependent fluorinases / chlorinases, SAM hydroxide transferases, etc. They mainly came from Bacteroidota (26.37%), Bacillota (21.06%), and Campylobacterota (19.20%). Based on sequence similarity, they could be divided into 914 families. After performing sequence-structure-function analyses such as sequence conservation analysis, homology modeling, and molecular docking on the families with higher alignment scores, diversity analysis was carried out on the constructed potential fluorinase database, and multiple potential fluorinase candidate clusters were screened out. First, sequences with a rare codon ratio exceeding 15% or containing transmembrane domains were removed to reduce the possibility of failed heterologous expression; second, sequences compatible with the Escherichia coli expression system were preferentially considered (such as moderate GC content and no toxicity tag prediction); finally, sequences with accessible active pockets and no significant steric hindrance were selected.

[0043] According to the above principles, the original sequence of S-adenosyl-L-methionine hydroxide transferase (WP_158858558.1) was screened and obtained. After codon optimization, the nucleotide sequence was as shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein was as shown in SEQ ID NO.2. It was handed over to a biological company to synthesize the recombinant plasmid pET-28a(+)-Flu-3 dry powder containing the target gene Flu-3, and the insertion sites were NdeI and XhoI.

[0044] SEQ ID NO.1

[0045] atgaataaac gcttacatct tgttttgttt ctttttgttg tatgcttgca ggtgcgagcc 60 caaaatcagg tcttggtgtt tcagtcagat tttggtacca aagatggggc ggtagcggcc 120 atgaaagggg ttgccgtggg cctgtccagt gacctgaaaa tatttgatct tacccatgaa 180 attccgccgt tcgatatttg ggaagctgct tataggctgg agcaaacggc ggagtattgg 240 cctgccggaa ccgtatttgt atccgtggtg gaccccggtg tgggaagcga acgtaaatct 300 gtcgtgctga agaccaagag cgggcacttt tttgtcaccc ctgacaatgg cactctgacg 360 ctggttgcgg aaacgctagg aatagaacag gtaaggggga ttgatgaagc aaagaaccgc 420 cgtgcaaatt ccgaaaagtc atacaccttt cacggaaggg atgtttacgc ctacacaggg 480 gcggcgcttg cctccggaaa aatatctttt gaggaggtag gaggggaact tcccgcaaaa 540 attgtagtcc taccctacga aagaccccga atggaaggcg gtgtgattag aggcaatatc 600 atcattctag atccgcagta tggcaatatt tggacaaata ttcaccagtc actggtggaa 660 aagctaaacc taagctacgg agatttgttg aaagttagta taatttatca cgatagaatc 720 gttttcgatg aagaagttcc tttttcaaaa acctttgagg atgtgggggt tggtgagaat 780 ttggcgtatc ttaacagtct gctgaatttt tccctggcca tcaatcaggg caacttcgct 840 acagcatatg aaataaaaag tggcgaaggt tggaaaatcc agctaaagcc agccaagtaa 900

[0046] Dissolve 4 μg of the above-mentioned recombinant plasmid dry powder in 40 μL of ultrapure water, mix it with 100 μL of competent E. Coli BL21(DE3), place it on ice for 20 - 30 min, immediately take it out after heat shock at 42 °C for 90 s and then place it on ice for 5 min, add 800 μL of antibiotic-free LB liquid medium, resuscitate at 37 °C and 200 rpm for 1 h, centrifuge, discard 800 μL of the supernatant, mix the remaining liquid with the precipitate and then spread it on an LB solid medium plate containing 30 μg / mL kanamycin, culture overnight at 37 °C, pick single colonies to extract the recombinant plasmid for sequencing verification, screen to obtain the positive transformant E. coli BL21(DE3)-pET-28a(+)-Flu-3, and store it in a glycerol tube.

[0047] 2. Induced expression of the enzyme and preparation of crude enzyme solution

[0048] Take out the glycerol storage tube of the above-mentioned E. coli BL21(DE3)-pET-28a(+)-Flu-3, after natural melting, streak the bacterial solution on an LB solid medium plate containing 30 μg / mL kanamycin resistance, invert and culture overnight at 37 °C. Pick a single colony and inoculate it into a test tube containing 30 μg / mL kanamycin-resistant LB liquid medium, shake culture overnight at 37 °C and 200 rpm to obtain the activated seed solution. Inoculate the activated seed solution into a 100 mL shake flask containing 30 μg / mL kanamycin-resistant LB liquid medium at an inoculation amount of 2% (v / v), shake culture at 37 °C and 200 rpm for about 2 h until OD 600 = 0.6 - 0.8. Add IPTG with a final concentration of 0.1 mM, induce by shaking at 18 °C and 120 rpm for 18 - 24 h. Centrifuge the induced bacterial solution at 4 °C and 3500 rpm for 20 min using a vertical large-capacity centrifuge, discard the supernatant, dissolve the wet bacterial cells in 15 mL of PBS and resuspend the cells. Perform ultrasonic disruption on the cell suspension, disrupt at a power of 40 W for 15 min until the solution becomes clear, with a disruption time of 3 s and an interval of 5 s. Gently mix the sample with a shaker, then centrifuge the sample at 4 °C and 12000 rpm for 30 min, collect the supernatant and precipitate, and the supernatant is the crude enzyme solution.

[0049] 3. Purification of the enzyme

[0050] Select an appropriate amount of Ni-NTA affinity resin and load it into a suitable chromatography column (15.6×79.2 mm). The loading amount of the affinity resin is 1 / 5 column volume. Subsequently, perform three washing operations with pure water (15 mL). Then, perform two equilibration treatments with NTA-0 eluent (15 mL), and also let it stand after each equilibration. This is the pretreated Ni-NTA resin. Composition of NTA-0 eluent: 20 mM Tris, 500 mM NaCl, 10% (v / v) glycerol, and the solvent is water.

[0051] Mix 30 mL of the above crude enzyme solution with the pretreated Ni-NTA resin (3 mL) in a small beaker, and use an oscillating thermostatic metal bath device to chelate at 4°C for 3 h. Inject the chelated mixture into the chromatography column and collect the effluent, which is the protein flow-through solution. Subsequently, perform elution operations successively with NTA-20 eluent (10 mL), NTA-50 eluent (5 mL), NTA-100 eluent (15 mL), and NTA-300 eluent (15 mL) according to the preset gradient. The eluent needs to be collected using 10 mL EP tubes, with 5 mL collected in each tube. Verify the tube number and corresponding purity of the target protein by SDS-PAGE detection, as Figure 1 shown. Collect the target protein according to the SDS-PAGE results, and obtain the pure enzyme solution of S-adenosyl-L-methionine hydroxide transferase after ultrafiltration and concentration to remove salts using a 3 KDa ultrafiltration tube.

[0052] NTA-20 eluent: 20 mM Tris, 500 mM NaCl, 10% (v / v) glycerol, 20 mM imidazole, and the solvent is water.

[0053] NTA-50 eluent: 20 mM Tris, 500 mM NaCl, 10% (v / v) glycerol, 50 mM imidazole, and the solvent is water.

[0054] NTA-100 eluent: 20 mM Tris, 500 mM NaCl, 10% (v / v) glycerol, 100 mM imidazole, and the solvent is water.

[0055] NTA-300 eluent: 20 mM Tris, 500 mM NaCl, 10% (v / v) glycerol, 300 mM imidazole, and the solvent is water.

[0056] 4. Determination of protein concentration of the enzyme

[0057] In the examples of the present invention, the protein concentration is measured using a BCA protein concentration assay kit.

[0058] Take an appropriate amount of 25 mg / mL protein standard solution and dilute it to 0.5 mg / mL with PBS buffer. Add the standard products in volumes of 0, 1, 2, 4, 8, 12, 16, and 20 μL to the standard product wells of a 96-well plate, and supplement with standard product diluent to make up to 20 μL.

[0059] Take an appropriate amount of pure S-adenosyl-L-methionine hydroxide transferase solution, dilute it 10-fold with PBS buffer, and then take 20 μL and add it to the sample wells of a 96-well plate.

[0060] According to the number of standard products and samples, prepare an appropriate amount of BCA working solution by mixing BCA kit solution A and BCA kit solution B at a volume ratio of 50:1, and mix well. Add 200 μL of BCA working solution to each standard product well and sample well, and incubate at 37 °C for 30 min. Measure the absorbance of each well at 562 nm using an enzyme-linked immunosorbent assay (ELISA) reader, plot a standard curve, calculate the protein concentration in the sample based on the standard curve, and dilute it to 5 mg / mL.

[0061] Example 2: 5'-substitution reaction catalyzed by S-adenosyl-L-methionine hydroxide transferase Flu-3 with S-adenosylmethionine as the substrate

[0062] Experimental group: Add the pure S-adenosyl-L-methionine hydroxide transferase Flu-3 solution with a protein concentration of 5 mg / mL prepared in Example 1 to 50 mM Tris-HCl buffer (pH = 8.0), and then add 5 μL of 12 mM substrate S-adenosyl-L-methionine solution (the solvent is 50 mM Tris-HCl buffer (pH = 8.0)) to form a 100 μL reaction system, so that the final concentration of the added pure enzyme solution is 0.5 mg / mL in terms of protein content, and the final concentration of the added substrate is 600 μM. React at 37 °C for 1 h. After the reaction, heat the reaction solution in a boiling water bath for 5 min, centrifuge to remove denatured proteins, and perform HPLC analysis and LC-MS analysis on the supernatant.

[0063] Product control group: Add the product adenosine to 50 mM Tris-HCl buffer (pH = 8.0) to form a 100 μL reaction system, and the final concentration of the added product is 600 μM. React at 37 °C for 1 h. After the reaction, heat the reaction solution in a boiling water bath for 5 min, centrifuge to remove denatured proteins, and perform HPLC analysis on the supernatant.

[0064] Substrate control group: Add the substrate SAM to 50 mM Tris-HCl buffer (pH = 8.0) to form a 100 μL reaction system, and the final concentration of the added substrate is 600 μM. React at 37 °C for 1 h. After the reaction, heat the reaction solution in a boiling water bath for 5 min, centrifuge to remove denatured proteins, and perform HPLC analysis on the supernatant.

[0065] Enzyme control group: The pure enzyme solution of S-adenosyl-L-methionine hydroxide transferase Flu-3 with a protein concentration of 5 mg / mL prepared in Example 1 was added to 50 mM Tris-HCl buffer (pH = 8.0) to form a 100 μL reaction system. The final concentration of the added pure enzyme solution was 0.5 mg / mL based on the protein content. The reaction was carried out at 37 °C for 1 h. After the reaction, the reaction solution was heated in a boiling water bath for 5 min, and the denatured proteins were removed by centrifugation. The supernatant was subjected to HPLC analysis.

[0066] The HPLC analysis conditions were as follows: Shimadzu liquid chromatograph SPD-20 was used, with a Waters X Select HSS T3 chromatographic column (150×4.6 mm, 5 μm). The detection wavelength was 254 nm, and the mobile phase was an acetonitrile-water system (containing 0.1% TFA (trifluoroacetic acid)). From 0 to 5 min (5% acetonitrile), from 5 to 12 min (5% - 95% acetonitrile), from 12 to 22 min (95% acetonitrile), from 22 to 25 min (95% - 5% acetonitrile), from 25 to 26 min (5% acetonitrile), the flow rate was 0.5 mL / min, the column temperature was 40 °C, and the injection volume was 10 μL.

[0067] The LC-MS (ESI-Q-TOF) detection conditions were as follows: A high-resolution mass spectrometer SCIEX X500B QTOF was used, with an Agilent poroshell 120SB-C18 UPLC reversed-phase column (2.1×50 mm, particle size 1.9 μm). The flow rate was 0.3 mL / min, the injection volume was 1 μL, and other methods were the same as the above liquid phase method.

[0068] The results are shown in Figure 3 As shown, compared with different control groups, the experimental group's S-adenosyl-L-methionine hydroxide transferase Flu-3 catalyzed the substrate to produce the hydroxylated product adenosine.

[0069] Example 3: Enzymatic properties of S-adenosyl-L-methionine hydroxide transferase

[0070] 1. Adenosine standard curve:

[0071] Six standard samples with concentrations of 10 μM, 20 μM, 50 μM, 80 μM, 100 μM, 200 μM, and 600 μM were prepared using adenosine standard products with pure water respectively. Then, through the HPLC detection described in Example 2, the peak area sizes corresponding to the concentrations of each standard product were obtained. A standard curve was made with the peak area as the ordinate and the concentration as the abscissa. The results are shown in Figure 4 , and the results show that within the tested concentration range, there is a good linear relationship between the concentration of adenosine and the peak area measured by HPLC, and this equation can be used to estimate the concentration of unknown samples.

[0072] 2. Effect of pH on enzyme activity

[0073] Take 10 μL of the pure enzyme solution prepared in Example 1 (protein concentration 5 mg / mL) and 5 μL of an aqueous solution of S-adenosyl-L-methionine (12 mM), and add 85 μL of buffer solutions with different pH values: 50 mM acetic acid-sodium acetate buffer (pH 4.0 - 5.0), 50 mM potassium phosphate buffer (pH 6.0 - 7.0), 50 mM Tris-HCl buffer (pH 7.5 - 8.5), 50 mM glycine-sodium hydroxide buffer (pH 9.0 - 10.0) to form a 100 μL reaction system. Incubate at 37 °C for 1 h. After the reaction is completed, heat the reaction solution at 100 °C for 5 min to inactivate the enzyme by denaturation, then centrifuge to collect the supernatant, and detect it using the HPLC described in Example 2. Calculate its concentration according to the adenosine standard curve based on the product peak area. Calculate the activity at different pH values based on the amount of product generated, take the maximum activity as 100%, and then calculate the relative activity at the remaining pH values. The results are shown in Figure 5 , and the results show that pH has a great influence on the activity of this enzyme. The enzyme has relatively high activity under alkaline conditions (pH around 7.0 - 10.0), its activity decreases significantly in the range of pH 4.0 - 6.5, and it has the best activity at pH 8.0.

[0074] Definition of enzyme activity: Under the specified temperature and pH conditions, the amount required to catalyze the formation of 1 μmol of product per unit time is 1 enzyme activity unit U. Take the maximum enzyme activity (the highest value in the test conditions) as 100%, and the activity under other conditions is the percentage relative to the maximum value.

[0075] 3. Effect of temperature on enzyme activity

[0076] Take 10 μL of the pure enzyme solution prepared in Example 1 (protein concentration 5 mg / mL) and 5 μL of an aqueous solution of S-adenosyl-L-methionine (12 mM), and add them to 85 μL of 50 mM Tris-HCl buffer (pH = 8.0) to form a 100 μL reaction system. Incubate at different temperatures (20 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C) for 1 h. After the reaction is completed, heat the reaction solution in a 100 °C metal bath for 5 min to inactivate the enzyme by denaturation, then centrifuge to collect the supernatant, and detect it using the HPLC described in Example 2. Calculate its concentration according to the adenosine standard curve based on the product peak area. Calculate the activity at different temperatures based on the amount of product generated, take the maximum activity as 100%, and then calculate the relative activity at the remaining temperatures. The results are shown in Figure 6 , and the results show that the enzyme purified in this experiment has the highest activity at 30 °C, and its activity gradually decreases as the temperature gradually increases. Even at 80 °C, the enzyme can still retain about 50% of its activity.

[0077] 4. Effects of Different Metal Ions on Enzyme Activity

[0078] Take 10 μL of the pure enzyme solution prepared in Example 1 (protein concentration 5 mg / mL), 5 μL of an aqueous solution of S-adenosyl-L-methionine (12 mM), add them to 84 μL of 50 mM Tris-HCl buffer (pH = 8.0), and then add 1 μL of aqueous solutions of different metal ions (Na + , K + , Mg 2+ , Zn 2+ , Mn 2+ , Cu 2+ , Co 2+ , EDTA, Ca 2+ ) to make their final concentrations all 1 mM to form a 100 μL reaction system, and incubate at 37 °C for 1 h. After the reaction is completed, heat at 100 °C for 5 min, centrifuge to collect the supernatant, and detect it using the HPLC described in Example 2. Calculate its concentration according to the adenosine standard curve based on the product peak area. Calculate the effects of different metal ions on enzyme activity according to the amount of the generated product. Take the activity of the control group as 100%, and then calculate the relative activities under the remaining experimental groups. The results are shown in Figure 7 , and the results show that when EDTA is added, the enzyme activity increases, while when Cu 2+ , Ca 2+ and Na + are added, the enzyme activity is significantly inhibited, indicating that the enzyme activity may depend on specific metal ions and is significantly inhibited by Cu 2+ , Ca 2+ and Na + .

[0079] 5. Relative Activity of the Enzyme at Different Incubation Times at 25 °C

[0080] Divide the pure enzyme solution prepared in Example 1 into 5 tubes, 10 μL per tube, and incubate them at 25 °C in a constant temperature incubator for different times: 0, 1, 2, 3, 4 days. Set 0 day as Control, and the others as experimental groups. Add 5 μL of an aqueous solution of S-adenosyl-L-methionine (12 mM) to each, and add 85 μL of 50 mM Tris-HCl buffer (pH = 8.0) to form a 100 μL reaction system, and react at 37 °C for 1 h. After the reaction is completed, inactivate in a boiling water bath for 5 min, centrifuge to collect the supernatant, and detect it using the HPLC described in Example 2. Calculate its concentration according to the adenosine standard curve based on the product peak area. Detect whether the enzyme activity is affected when the enzyme is placed at room temperature for different times according to the amount of the generated product. Take the activity at 0 day as 100%, and then calculate the remaining relative activities. The results are shown in Figure 8, The results showed that as the number of days of storage increased, although the enzyme activity gradually decreased, the degree of decrease was small, and the enzyme activity was still about 78% on the fourth day of storage. This indicated that the protein purified by us had good stability.

[0081] Example 4: Activity Assay of S-adenosyl-L-hydroxide Transferase

[0082] Take 10 μL (5 mg / mL) of the pure enzyme solution (Flu-3) prepared in Example 1 and 5 μL (12 mM) of S-adenosyl-L-methionine aqueous solution, and add them to 85 μL of 50 mM Tris-HCl buffer (pH = 8.0) to form a 100 μL reaction system, and incubate at 37 °C for 1 hour. Set 3 parallels.

[0083] In addition, take 10 μL (5 mg / mL) of the pure enzyme solution of another S-adenosyl-L-hydroxide transferase Flu-5 that has been mined and heterologously expressed, and incubate at 37 °C for 1 hour under the same above conditions.

[0084] After the reaction ended, heat the reaction solution in a 100 °C metal bath for 5 min to inactivate the enzyme by denaturation, then centrifuge to collect the supernatant, and detect it using the HPLC described in Example 2. Calculate its concentration according to the adenosine standard curve based on the product peak area. Calculate the activity under different enzyme reactions according to the amount of the generated product, and take the maximum activity as 100%. The results showed that under the conditions of pH = 8.0 and 37 °C, the yield of the product catalyzed by the enzyme Flu-3 purified in the present invention was about 22.0% higher than the activity of another screened S-adenosyl-L-hydroxide transferase Flu-5 per unit time, and the amount of the product catalyzed by the enzyme per minute in each milliliter of the reaction system increased by 0.0028 μmol.

[0085] Table 1 Comparison of Enzyme Activities

[0086]

Claims

1. An S-adenosyl-L-methionine hydroxide transferase, characterized in that, The amino acid sequence of the S-adenosyl-L-methionine hydroxide transferase is shown in SEQ ID NO.

2.

2. A recombinant genetically engineered bacterium containing the coding gene of the S-adenosyl-L-methionine hydroxide transferase described in claim 1.

3. Use of the S-adenosyl-L-methionine hydroxide transferase described in claim 1 in the preparation of adenosine from S-adenosylmethionine.

4. The application according to claim 3, characterized in that The said use is as follows: Using the pure enzyme solution extracted after ultrasonic disruption of the wet bacterial cells obtained by induced culture of the recombinant genetically engineered bacterium containing the coding gene of S-adenosyl-L-methionine hydroxide transferase as the catalyst, using S-adenosylmethionine as the substrate, and using a buffer solution with pH 6 - 10 as the reaction medium to form a reaction system. After the reaction is completed at 20 - 60 °C, the reaction solution is separated and purified to obtain adenosine.

5. The application according to claim 4, characterized in that In the said reaction system, the final concentration of the substrate added is 300 - 900 μM, and the final concentration of the catalyst added is 0.1 - 5 mg / mL in terms of protein content.

6. The application according to claim 4, characterized in that, The reaction system also contains EDTA or metal ions, and the metal ions include Na + , K + , Mg 2+ , Zn 2+ , Mn 2+ , Cu 2+ , Co 2+ , Ca 2+ ; the final concentration of the added EDTA or metal ions is 0.1 - 0.5 mM.

7. The application according to claim 4, characterized in that, The said reaction is carried out at 25 - 30 °C for 1 h.

8. The application according to claim 4, wherein The said catalyst is prepared as follows: (1) Preparation of wet bacterial cells: Streak the glycerol stock solution of the recombinant genetically engineered bacterium containing the gene encoding S-adenosyl-L-methionine hydroxide transferase on an LB solid medium plate containing 30 μg / mL kanamycin resistance, and incubate it upside down at 37 °C overnight; pick a single colony and inoculate it into a test tube containing LB liquid medium with 30 μg / mL kanamycin resistance, and incubate it at 37 °C with shaking at 200 rpm overnight to obtain the activated seed solution; inoculate the activated seed solution into a shake flask containing LB liquid medium with 30 μg / mL kanamycin resistance at an inoculation amount of 2% (v / v), and incubate it at 37 °C with shaking at 200 rpm until OD 600 = 0.6 - 0.8, add IPTG with a final concentration of 0.1 mM, and induce it with shaking at 18 °C and 120 rpm for 18 - 24 h; centrifuge the induced bacterial solution at 4 °C and 3500 rpm for 20 min using a vertical large-capacity centrifuge, discard the supernatant, and collect the wet bacterial cells; (2) Preparation of crude enzyme solution: Resuspend the wet bacterial cells in PBS, perform ultrasonic disruption on the bacterial suspension, disrupt at a power of 40 W for 15 min until the solution becomes clear, with a disruption time of 3 s and an interval of 5 s. After gently mixing the sample with a shaker, centrifuge the sample at 4 °C and 12,000 rpm for 30 min, collect the supernatant and precipitate, and the supernatant is the crude enzyme solution; (3) Preparation of pure enzyme solution: Load Ni-NTA affinity resin into the chromatography column, wash it three times with pure water, with each elution volume being 3 column volumes; then, perform two equilibration treatments with NTA-0 eluent, with each volume being 3 column volumes, and let it stand after each equilibration, which is the pretreated Ni-NTA resin; The composition of NTA-0 eluent: 20 mM Tris, 500 mM NaCl, 10% glycerol, and the solvent is water; Mix the above crude enzyme solution with the pretreated Ni-NTA resin at a volume ratio of 10:1, and chelate at 4 °C for 3 h using an oscillating constant-temperature metal bath device; Inject the chelated mixture into the chromatography column, and sequentially elute with NTA-20 eluent, NTA-50 eluent, NTA-100 eluent, and NTA-300 eluent, with elution volumes of 2 / 3, 1 / 3, 1, and 1 column volumes respectively; Collect the effluent containing the target protein, and obtain the pure enzyme solution of S-adenosyl-L-methionine hydroxide transferase after ultrafiltration and concentration through a 3 KDa ultrafiltration tube to remove salts; NTA-20 eluent: 20 mM Tris, 500 mM NaCl, 10% glycerol, 20 mM imidazole, and the solvent is water; NTA-50 eluent: 20 mM Tris, 500 mM NaCl, 10% glycerol, 50 mM imidazole, and the solvent is water; NTA-100 eluent: 20 mM Tris, 500 mM NaCl, 10% glycerol, 100 mM imidazole, and the solvent is water; NTA-300 eluent: 20 mM Tris, 500 mM NaCl, 10% glycerol, 300 mM imidazole, with water as the solvent.

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