Solution and method used for enzyme synthesis of s-adenosylmethionine

Abstract

A solution containing a boric acid, an S-adenosylmethionine synthase, ATP, methionine, and water.

Description

Solutions and methods for enzymatic synthesis of S-adenosylmethionine

[0001] The present disclosure relates to a solution and a method for enzymatically synthesizing S-adenosylmethionine, and more particularly to a solution for enzymatically synthesizing S-adenosylmethionine, a method for increasing the amount of S-adenosylmethionine produced, a method for improving the activity of S-adenosylmethionine synthetase, and a method for producing S-adenosylmethionine.

[0002] S-Adenosylmethionine (SAM) is an important substance responsible for donating methyl groups in the body. In recent years, its usefulness has attracted attention for its use as a supplement.

[0003] The most common method for producing SAM is extraction from SAM-containing biological cells (Patent Document 1). Enzymatic synthesis of SAM has been considered unrealistic due to the low SAM productivity of S-adenosylmethionine synthase (SAM synthase).

[0004] The reason for the low SAM productivity of SAM synthase is thought to be that competitive inhibition occurs with the product, SAM (Non-Patent Document 1).

[0005] Regarding the release of competitive inhibition of SAM synthase, it has been reported that the presence of urea or the like dissociates the bond between SAM and SAM synthase, making it less susceptible to inhibition and contributing to improved activity (Non-Patent Document 2), but it could not be said that sufficient activity was obtained to be applicable to industrial enzyme synthesis.

[0006] SAM has two optical isomers, SS-SAM and RS-SAM, due to the stereoisomerism of the methyl group coordinated to the sulfur atom, and it is known that only SS-SAM has physiological activity as a methyl group donor (Non-Patent Documents 1 and 2). It has been reported that only SS-SAM is produced by SAM synthase (Non-Patent Documents 1 and 3).

[0007] SAM functions as a methyl group donor for various molecules in vivo, including the methylation of DNA and RNA, and is essential for reactions such as the m7Gppp modification (=Cap0 modification) of the 5' end of RNA by capping enzymes (Non-Patent Document 4).

[0008] Japanese Unexamined Patent Publication No. 48-80788

[0009] Park et al., “Enzymatic Synthesis of S-Adenosyl-L-methionine on the Preparative Scale”, Bioorganic & Medicinal Chemistry, Vol. 4, No. 12, pp. 2179-2185, 1996Matos JR and Wong C., “S-Adenosylmethionine: Stability and Stabilization”, Bioorganic Chemistry, vol, 15, pp. 71-80, 1987Chris RV and Steven GC, “Recognition of Age-damaged (R,S)-Adenosyl-L-methionine by Two Methyltransferases in the Yeast Saccharomyces cerevisiae”, The Journal of Biological Chemistry, Vol. 282, No. 12, pp. 8604-8612, 2007Ramanathan A. et al., “SURVEY AND SUMMARY mRNA capping: biological functions and applications” Nucleic Acids Research, Vol. 44, No. 16, pp. 7511-7526, 2016

[0010] An object of the present disclosure is to solve various problems in the enzymatic synthesis of SAM and to provide an industrial enzymatic synthesis method for SAM and high-quality SAM. That is, an object of the present disclosure is to provide an industrial enzymatic synthesis method that avoids product inhibition of SAM synthase and achieves high SAM productivity. Specifically, an object of the present disclosure is to provide a solution to be used in an industrial enzymatic synthesis method that avoids product inhibition of SAM synthase and achieves high SAM productivity, a method for increasing the amount of S-adenosylmethionine produced, a method for improving the activity of S-adenosylmethionine synthase, and a method for producing S-adenosylmethionine.

[0011] The present disclosure was completed as a result of intensive research conducted to solve the above-mentioned problems, and the discovery that adding boric acids to the reaction system of SAM synthase relieves product inhibition of SAM synthase and improves enzyme activity.

[0012] That is, the present disclosure relates to a solution containing boric acids, S-adenosylmethionine synthase, ATP, methionine, and water.

[0013] The present disclosure also relates to a method for increasing the amount of S-adenosylmethionine produced, which comprises adding boric acid to a solution containing S-adenosylmethionine synthase, ATP, methionine, and water.

[0014] The present disclosure also relates to a method for improving the activity of S-adenosylmethionine synthetase, which comprises adding boric acid to a solution containing S-adenosylmethionine synthetase, ATP, methionine, and water.

[0015] Furthermore, the present disclosure relates to a method for producing S-adenosylmethionine using an S-adenosylmethionine synthetase, the method comprising the step of producing S-adenosylmethionine in a solution containing S-adenosylmethionine synthetase, ATP, methionine, water, and boric acids.

[0016] The present disclosure provides an industrial enzymatic synthesis method that avoids product inhibition of SAM synthase, activates SAM synthase, and achieves high SAM productivity. Specifically, the present disclosure provides a solution used in the industrial enzymatic synthesis method that avoids product inhibition of SAM synthase and achieves high SAM productivity, a method for increasing the amount of S-adenosylmethionine produced, a method for improving the activity of S-adenosylmethionine synthase, and a method for producing S-adenosylmethionine.

[0017] 1 is a graph showing the results of Experimental Example 2. The vertical axis shows the SAM synthesis activity (units / mL) calculated from the amount of SAM produced. The horizontal axis shows the type of sample, with the left side showing samples without boric acid and the right side showing samples with boric acid added. This is a graph showing the results of Experimental Example 3. The horizontal axis shows 0, 1, 2, 3, 4, and 5 hours, which are the elapsed time from the start of the reaction when a portion of the reaction solution was taken. The vertical axis shows the amount of SAM produced (mM). Urea (+), boric acid (+) refers to a sample to which 2 M urea and 100 mM boric acid were added. Urea (-), boric acid (+) refers to a sample to which 100 mM boric acid was added. Urea (+), boric acid (-) refers to a sample to which 2 M urea was added. Urea (-), boric acid (-) refers to a sample to which neither urea nor boric acid was added. This is a graph showing the results of Experimental Example 4. The horizontal axis indicates the elapsed time from the start of the reaction when an aliquot of the reaction solution was taken, at 0, 1, 2, and 3 hours. The vertical axis indicates the amount of SAM produced (mM). "Substrate 30 mM" refers to a sample to which the substrates ATP and L-methionine were added to a final concentration of 30 mM, and "Substrate 60 mM" refers to a sample to which the substrates ATP and L-methionine were added to a final concentration of 60 mM. This is a graph showing the results of Experimental Example 6. The vertical axis indicates the amount of Cap0 produced per hour (pmol / min). The horizontal axis indicates the type of sample; the left side indicates a sample using a SAM according to an embodiment of the present invention as a raw material, and the right side indicates a sample using a commercially available SAM as a raw material.

[0018] The following is a detailed description of the embodiments of the present invention. The present invention is not limited to the following embodiments, and it should be understood that modifications and improvements made to the following embodiments based on the ordinary knowledge of those skilled in the art without departing from the spirit of the present invention are also within the scope of the present invention.

[0019] In this specification, unless otherwise specified, a numerical range expressed using "to" means a range that includes the numerical values ​​written before and after "to" as the lower and upper limits. In addition, in this specification, when a numerical value is followed by "more than" or "less than," the numerical range means a range that does not include the numerical value as the lower or upper limit. In addition, for numerical ranges described in stages in this specification, the upper limit of a certain numerical range may be replaced with the upper limit of another numerical range described in stages or a value shown in an example. In addition, for numerical ranges described in stages in this specification, the lower limit of a certain numerical range may be replaced with the lower limit of another numerical range described in stages or a value shown in an example. Furthermore, a numerical range may be a combination of any of the upper and lower limit values ​​described in this specification. In addition, "%" in this specification is based on mass unless otherwise specified.

[0020] An embodiment of the present invention relates to activation of a SAM synthase by boric acids and a method for enzymatically synthesizing SAM using the same.

[0021] In this specification, the term "boric acids" refers to compounds that function as Lewis bases consisting of a boron atom and an atom with strong electronegativity such as oxygen or fluorine, or compounds that function as Lewis bases in which an organic functional group is coordinated to boric acid, such as tetrakis(4-chlorophenyl)boric acid. In this specification, the term "boric acids" also includes salts of the above-mentioned boric acids.

[0022] As used herein, S-adenosylmethionine (S-adenosylmethionine, S-(5'Adenosyl)-L-methionine, SAM) refers to the optical isomer (SS)-S-adenosyl-L-methionine of the compound designated by CAS Registry Number 29908-03-0. As used herein, (RS)-S-adenosyl-L-methionine, an optical isomer of S-adenosylmethionine, is distinguished from S-adenosylmethionine. As used herein, when describing a concept encompassing (SS)-S-adenosyl-L-methionine and (RS)-S-adenosyl-L-methionine, the term SAM (a mixture of SS-SAM and RS-SAM) is used for the purpose of distinction. As used herein, the term "S-adenosylmethionine" also encompasses salts of S-adenosylmethionine, which may be hydrochlorides, tosylate salts, sulfate salts, phosphate salts, or any other anionic salts.

[0023] In this specification, when the molar concentration of S-adenosylmethionine is described, unless otherwise specified, it refers to the molar extinction coefficient (SAM) of 15,400 M as described in Matos JR and Wong C. "S-adenosylmethionine: Stability and stabilization", Bioorganic Chemistry, Vol. 15, Issue 1, pp. 71-80, 1987. -1 Use the value calculated from the above.

[0024] As used herein, S-adenosylmethionine synthase (methionine-adenosyltransferase, SAM synthase) refers to an enzyme defined in EC 2.5.1.6. S-adenosylmethionine synthase defined in EC 2.5.1.6 has the following activity: ATP + L-Methionine + HO <=> Orthophosphate + Diphosphate + S-Adenosyl-L-methionine As used herein, the term "S-adenosylmethionine synthase activity" refers to the activity of catalyzing a reaction that produces S-adenosylmethionine from one molecule of ATP, one molecule of L-methionine, and one molecule of water in the presence of S-adenosylmethionine synthase, ATP, L-methionine, and a water molecule.

[0025] Unless otherwise specified, enzyme activity in this specification is measured using an enzyme solution prepared by a conventional method under the following conditions: The enzyme solution is added to a mixed solution containing 30 mM magnesium chloride, 10 mM methionine, 10 mM ATP, 100 mM Tris-HCl buffer (pH 8.0), and 100 mM KCl, and the reaction is carried out by incubating at 37°C for 30 minutes, followed by heat treatment at 100°C for 30 seconds to terminate the reaction.

[0026] In the activity measurement herein, the amount of substances such as S-adenosylmethionine contained in the reaction solution obtained by the above method is quantified using HPLC. The HPLC conditions are as follows. A Triart C18 column (particle size 3 μm, inner diameter 2.0 mm, length 150 mm) manufactured by YMC Corporation is used. The mobile phase is 50 mM ammonium phosphate, 1% trifluoroacetic acid (pH 5.4), with a flow rate of 0.3 mL / min and room temperature. For detection, 10 μL of a sample prepared by diluting the reaction solution 50-fold with the mobile phase is applied to the column, and absorbance at a wavelength of 260 nm is measured. Under the above measurement conditions, SS-SAM shows a retention time of 16.5 minutes, and RS-SAM shows a retention time of 18.0 minutes.

[0027] As used herein, the term "1 U of S-adenosylmethionine synthetase" refers to the activity of producing 1 μmol of SAM per minute at 37° C. in the presence of methionine and ATP.

[0028] As used herein, the phrase "high enzymatic activity of S-adenosylmethionine synthase" refers to high activity of the enzyme to synthesize S-adenosylmethionine. As used herein, the phrase "high enzymatic activity" without specifying the activity of the enzyme may refer to high activity to synthesize S-adenosylmethionine.

[0029] As used herein, the phrase "improving the activity of S-adenosylmethionine synthetase" means improving the activity of the enzyme to synthesize S-adenosylmethionine.

[0030] As used herein, the term "competitive inhibition of S-adenosylmethionine synthetase" means that the reaction of S-adenosylmethionine synthesis catalyzed by S-adenosylmethionine synthetase is inhibited by the presence of the product S-adenosylmethionine. As used herein, the term "competitive inhibition" without specifying the activity of any enzyme may mean that the reaction of S-adenosylmethionine synthesis catalyzed by S-adenosylmethionine synthetase is inhibited by the presence of the product S-adenosylmethionine.

[0031] As used herein, the phrase "removal of competitive inhibition of S-adenosylmethionine synthetase" means that the presence of the product S-adenosylmethionine does not result in feedback inhibition of the S-adenosylmethionine-producing reaction catalyzed by S-adenosylmethionine synthetase. In other words, the phrase "removal of competitive inhibition of S-adenosylmethionine synthetase by the addition of boric acids" means that the concentration of S-adenosylmethionine is increased when the enzyme reaction reaches equilibrium in the presence of boric acids, compared to the concentration of S-adenosylmethionine when the enzyme reaction reaches equilibrium in a general enzyme reaction without the addition of boric acids.

[0032] As used herein, urea is the compound designated by CAS Registry Number 57-13-6.

[0033] As used herein, "homology" generally refers to the percentage of identical amino acids in two or more amino acid sequences, calculated according to methods known in the art. Before calculating the percentage, the amino acid sequences to be compared are aligned, and gaps are introduced into the amino acid sequences, if necessary, to maximize the percentage of identity. Alignment methods, percentage calculation methods, comparison methods, and related computer programs are well known in the art (e.g., BLAST, GENETYX, etc.). Unless otherwise specified, "homology" as used herein can be expressed as a value measured by BLAST from NCBI (http: / / www.ncbi.nlm.nih.gov / ). When comparing amino acid sequences using BLAST, Blastp can be used with default settings as the algorithm. The measurement results are quantified as positives or identities.

[0034] As used herein, the term "vector" may refer to, for example, a plasmid derived from Escherichia coli (e.g., pBR322, pUC12, pET-Blue-2), a plasmid derived from Bacillus subtilis (e.g., pUB110, pTP5), a yeast-derived plasmid (e.g., pSH19, pSH15), an animal cell expression plasmid (e.g., pA1-11, pcDNAI / Neo), a bacteriophage such as λ phage, or a vector derived from a virus such as an adenovirus, retrovirus, or baculovirus. These vectors may contain components necessary for protein expression, such as a promoter, an origin of replication, or an antibiotic resistance gene. The vector may be an expression vector.

[0035] In one aspect, an embodiment of the present invention is a solution containing boric acids, a SAM synthase, ATP, methionine, and water. The coexistence of the boric acids and the SAM synthase increases the enzymatic activity of the SAM synthase, thereby improving SAM production capacity.

[0036] Another aspect of an embodiment of the present invention is a method for increasing the amount of SAM produced, comprising adding boric acid to a solution containing a SAM synthase, ATP, methionine, and water.

[0037] Another aspect of an embodiment of the present invention is a method for improving the activity of a SAM synthase, the method comprising adding boric acid to a solution containing a SAM synthase, ATP, methionine, and water.

[0038] Another aspect of the present invention is a method for producing SAM using a SAM synthase, comprising a solution containing a SAM synthase, ATP, methionine, water, and boric acids. Because this production method involves enzymatic synthesis of SAM, it is possible to specifically synthesize physiologically active SS-SAM. Because the SAM obtained by this production method has a high content of physiologically active SS-SAM, when the SAM is used as a raw material for a capping enzyme, the methyl group-donating ability of the capping enzyme is improved.

[0039] Examples of the boric acid used in the embodiment of the present invention include, but are not limited to, boric acid, metaboric acid, tetraboric acid, and tetrafluoroboric acid. The boric acid used in the embodiment of the present invention is not particularly limited, but from the viewpoints of availability, ease of handling, and solubility in water, boric acid, potassium tetraborate tetrahydrate, and sodium tetraborate (borax) are preferred, and potassium tetraborate tetrahydrate is more preferred.

[0040] There is no particular limitation on the origin of the boric acid used in the embodiment of the present invention, and commercially available boric acids may be purchased and used as appropriate.

[0041] The concentration of the boric acid may be selected as appropriate, but is exemplified as 0.01 to 0.20 mol / L per 1 U of SAM synthase. In particular, when potassium borate is used as the boric acid, considering its solubility in water, the concentration is preferably 0.03 to 0.18 mol / L, and more preferably 0.05 to 0.15 mol / L.

[0042] There is no limitation on the origin of the SAM synthase used in embodiments of the present invention, as long as it has the following activity defined in EC 2.5.1.6: ATP + L-Methionine + HO <=> Orthophosphate + Diphosphate + S-Adenosyl-L-methionine The reason there is no limitation on the origin of the SAM synthase used in embodiments of the present invention is that a hypothesis for the mechanism of action by which embodiments of the present invention are effective is that a radical possessed by SAM coordinates to the unpaired electron of boric acid, thereby efficiently releasing it from the active center of the SAM synthase, thereby relieving product inhibition, and therefore it is expected that any SAM synthase will be effective.

[0043] Examples of SAM synthases used in embodiments of the present invention include those derived from Escherichia coli, yeast, and cultured cells. Based on the results of the Examples described below, it is predicted that the addition of boric acid will improve SAM production, so the homology with the SAM synthase derived from Escherichia coli is preferably 50% or more, more preferably 60% or more, and even more preferably 70% or more. Furthermore, because the addition of boric acid is strongly predicted to improve SAM production, it is even more preferable that the homology with the SAM synthase derived from Escherichia coli is 80% or more, and particularly preferably 90% or more.

[0044] Examples of methods for obtaining the SAM synthase used in embodiments of the present invention include a method for obtaining a processed product of microbial cells that express the SAM synthase by genetic recombination and / or endogenously as a crude enzyme solution, or a method for obtaining the purified enzyme from the processed product. Microbial cells can be prepared by culturing the microorganisms in a medium in which they can grow using a conventional method, and then collecting the cells by centrifugation or the like. Specifically, taking bacteria belonging to the genus Escherichia as an example, the culture medium may be a bouillon medium, an LB medium (1.0% (w / v) tryptone, 0.5% (w / v) yeast extract, 0.5% (w / v) salt), or a 2xYT medium (1.6% (w / v) tryptone, 1.0% (w / v) yeast extract, 0.5% (w / v) salt), and after inoculating the seed culture medium, the culture is cultured at 30 to 50°C for about 1 to 100 hours with stirring as necessary, and the resulting culture medium is centrifuged to recover the microbial cells.

[0045] Examples of crude enzyme solutions include supernatants obtained by treating the above-mentioned microbial cells according to common treatment methods such as mechanical disruption (using a Waring blender, French press, homogenizer, mortar, etc.), freeze-thawing, autolysis, drying (by freeze-drying, air-drying, etc.), enzymatic treatment (by lysozyme, etc.), ultrasonic treatment, chemical treatment (by acid, alkali, etc.), and then centrifuging the disrupted cells.

[0046] Examples of purified enzymes include those obtained by subjecting fractions having the desired enzyme activity from the above-mentioned treated bacterial cells to conventional enzyme purification procedures (salting out, isoelectric precipitation, organic solvent precipitation, dialysis, various types of chromatography, etc.).

[0047] The SAM synthase used in the embodiment of the present invention produces SAM from a substrate by causing a reaction in a reaction system consisting of a solution containing the enzyme.

[0048] The solution used in the embodiment of the present invention contains ATP (adenosine triphosphate) as a substrate. The concentration of ATP in the solution can be selected appropriately, but is exemplified as 0.01 to 0.50 mol / L. Among these, from the viewpoint of a concentration that does not cause substrate inhibition, 0.01 to 0.03 mol / L is preferred, and 0.01 to 0.20 mol / L is more preferred.

[0049] The concentration of ATP per 1 U of SAM synthase can be selected as appropriate, but is, for example, 0.01 to 0.50 mol / L. In particular, the concentration is preferably 0.01 to 0.30 mol / L, and more preferably 0.01 to 0.20 mol / L, in terms of not causing substrate inhibition.

[0050] The solution used in the embodiment of the present invention contains methionine as a substrate. The concentration of methionine in the solution can be selected appropriately, but is, for example, 0.01 to 0.50 mol / L. In particular, in consideration of the solubility of methionine in water, the concentration is preferably 0.01 to 0.30 mol / L, and more preferably 0.01 to 0.20 mol / L.

[0051] The concentration of methionine per 1 U of SAM synthase can be selected as appropriate, but is, for example, 0.01 to 0.50 mol / L. In particular, taking into consideration the solubility of methionine in water, the concentration is preferably 0.01 to 0.30 mol / L, and more preferably 0.01 to 0.20 mol / L.

[0052] In an embodiment of the present invention, the quantitative ratio of ATP to methionine in the solution can be selected as appropriate. However, because one molecule of SAM is generated from one molecular equivalent of both, and because ATP is relatively unstable, it is preferable that the ATP (mol / L):methionine (mol / L) ratio be 1:1 to 2:1.

[0053] The solution used in the embodiment of the present invention contains water as a substrate. The concentration of water in the solution is not particularly limited and may be adjusted appropriately depending on the concentrations of other components.

[0054] The solution used in the embodiment of the present invention preferably further contains urea. Adding urea to the solution (reaction system) can additively improve the activity of the SAM synthase. The concentration of urea in the solution can be selected as appropriate, but is exemplified as 1.0 to 8.0 mol / L. In particular, from the viewpoint of maximizing the activity of the SAM synthase and considering the concentration range in which boric acids do not precipitate when coexisting with boric acids, a concentration of 1.2 to 5.0 mol / L is preferred, and a concentration of 1.5 to 3.0 mol / L is more preferred.

[0055] The concentration of urea per 1 U of SAM synthase can be selected as appropriate, but is exemplified as 1.0 to 8.0 mol / L. Among these, from the viewpoint of maximizing the activity of the SAM synthase and considering the concentration range in which boric acids do not precipitate when coexisting with boric acids, 1.2 to 5.0 mol / L is preferred, and 1.5 to 3.0 mol / L is more preferred.

[0056] The solution used in the embodiment of the present invention may contain other components as needed, including, but not limited to, metal salts (e.g., magnesium salts), buffers (e.g., Tris-HCl), reducing agents (e.g., dithiothreitol), etc.

[0057] The solution used in the embodiment of the present invention can undergo an enzymatic reaction under general temperature conditions. There are no particular limitations on the temperature at which the enzymatic reaction is carried out, and it can be set appropriately depending on the purpose. As an example of a suitable temperature condition, a temperature of 28°C or higher is preferred, 28°C to 79°C is more preferred, and 30°C to 70°C is even more preferred, as high enzymatic activity is demonstrated in the examples described below. Among these, a temperature of 37°C to 45°C is particularly preferred, as it exhibits high enzymatic activity.

[0058] The solution used in the embodiment of the present invention can undergo an enzymatic reaction under general pH conditions. There are no particular limitations on the pH at which the enzymatic reaction is carried out, and it can be set appropriately depending on the purpose. As an example of a suitable pH condition, a pH of 6.8 to 8.5 is preferred, as high activity has been demonstrated in the examples, and 8.0 is more preferred because it exhibits high enzymatic activity.

[0059] The SAM produced according to the present invention can be purified by a conventional method for purifying small molecules, such as, but not limited to, ion exchange purification, ultrafiltration, centrifugation, etc.

[0060] The following examples will be used to specifically explain the contents of the embodiments of the present invention, but the embodiments of the present invention should not be construed as being limited to these examples.

[0061] Experimental Example 1: Preparation of SAM synthase (1) Construction of plasmid DNA for overexpression of Escherichia coli SAM synthase A gene (metK, Genbank: BCA75389.1) encoding S-adenosylmethionine synthetase derived from Escherichia coli (Escherichia coli K12 strain) was artificially synthesized and inserted into the NcoI / BamHI restriction enzyme sites of the plasmid vector pTrc12-6 (JP 2001-103973 A) by infusion cloning. The constructed plasmid DNA (pTrc12-6-metK) was used to transform Escherichia coli JM109 strain, which was then cultured in a kanamycin-containing medium, and the plasmid DNA was amplified and purified.

[0062] (2) Preparation of SAM Synthase Solution: E. coli BL21 strain harboring the above-mentioned plasmid DNA (pTrc12-6-metK) was inoculated into 5 mL of LB medium (1.0% (w / v) peptone, 0.5% (w / v) yeast extract, 0.5% (w / v) sodium chloride) containing 50 μg / mL kanamycin and cultured at 37°C for 16 hours. 1 mL of the resulting preculture was inoculated into 50 mL of LBE medium (2.0% (w / v) potato peptone, 1.0% (w / v) yeast extract, 0.5% (w / v) sodium chloride) containing 50 μg / mL kanamycin and cultured with shaking at 37°C and 120 rpm. Isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 1.0 mM at an OD600 of approximately 0.5, and the culture was further cultured overnight (16 hours). After the incubation, the cells were collected by centrifugation (7,000 x g, 10 minutes), suspended in 5 mL of a buffer solution (50 mM potassium phosphate aqueous solution, pH 8.0), and then disrupted by ultrasonic treatment. The cell debris was removed by centrifugation (12,000 x g, 10 minutes), and the resulting supernatant was used as a SAM synthase solution.

[0063] Experimental Example 2: Examination of the effect of adding boric acid on improving SAM synthase activity The experiment in this example was carried out as follows. A mixture of 100 mM Tris-HCl (pH 8.0), 100 mM KCl, 30 mM magnesium chloride, 30 mM ATP, and 30 mM L-methionine was used as a control, and a mixture was prepared by adding 100 mM boric acid to the same mixture. A 10% amount of SAM synthase solution prepared by the method described in Experimental Example 1 was added to each reaction solution, and the reaction was carried out at 37°C. The reaction was stopped by heating at 100°C for 30 seconds, and the solution was diluted 50-fold with HPLC mobile phase and analyzed. SAM synthesis activity was calculated from the amount of SAM produced.

[0064] The results of the above experiment are shown in Figure 1. As shown in Figure 1, the SAM synthesis activity was higher in the case where boric acid was added (boric acid-containing condition) than in the case where boric acid was not added (control). Therefore, it was revealed that the addition of boric acid significantly increases the SAM synthesis activity compared to the case where boric acid was not added.

[0065] Experimental Example 3: Improvement of SAM Synthesis Amount by Combining Boric Acid with Urea, a Known Activator of SAM Synthesizing Enzymes. The experiment in this example was carried out as follows. A control mixture of 100 mM Tris-HCl (pH 8.0), 100 mM KCl, 30 mM magnesium chloride, 30 mM ATP, and 30 mM L-methionine was used. Mixtures were prepared by adding 100 mM boric acid, 2 M urea, or both to the control mixture. A 10% amount of SAM synthesizing enzyme solution prepared by the method described in Experimental Example 1 was added to each reaction solution, and the reaction was carried out at 37°C. Aliquots of the reaction solution were taken at 0, 1, 2, 3, 4, and 5 hours after the start of the reaction. The reaction solutions were diluted 50-fold with HPLC mobile phase and analyzed, and the amount of SAM produced was compared over time.

[0066] The results of the above experiment are shown in Figure 2. As shown in Figure 2, the amount of SAM synthesized was greater under both boric acid and urea addition conditions than under no addition conditions. Furthermore, by adding both boric acid and urea, approximately 15 mM SAM was produced at the 5-hour point. Because SAM synthase catalyzes an equilibrium reaction, the production of approximately 15 mM SAM in this reaction system using 30 mM ATP and 30 mM L-methionine as substrates can be said to indicate that inhibition of the enzyme reaction was relieved.

[0067] Experimental Example 4: Maximizing the amount of substrate in a SAM synthesis reaction Until now, in SAM synthesis using a SAM synthase, the maximum amount of substrate added was limited to 30 mM (Park et al., "Overcoming product inhibition of S-Adenosyl-L-methionine (SAM) synthetase: Preparation of SAM on the 30 mM scale", Bioorganic & Medicinal Chemistry Letters, Vol. 5, Issue 19, pp. 2203-2206, 1995). In the experiment of this example, it was verified whether the synthesis reaction of SAM synthase proceeds quantitatively without substrate inhibition or product inhibition even when the amount of substrate is increased, using a method for activating a SAM synthase according to an embodiment of the present invention.

[0068] The experiment in this example was carried out as follows. ATP and L-methionine were added to a solution containing 100 mM boric acid (pH 8.0), 100 mM potassium chloride, 30 mM magnesium chloride, 2 M urea, and 1.25 units / mL of SAM synthase to a final concentration of 30 mM or 60 mM (the ratio of ATP to L-methionine was the same for each final concentration), and the reaction was carried out at 37°C. Aliquots of the reaction solution were taken at 0, 1, 2, and 3 hours after the start of the reaction. The aliquots were analyzed using the HPLC analysis conditions described above.

[0069] The results of the above experiment are shown in Figure 3. As shown in Figure 3, at a substrate concentration of 30 mM, SAM was produced up to an equilibrium concentration of over 10 mM. It was revealed that due to the effect of eliminating inhibition of the SAM synthase according to an embodiment of the present invention, product inhibition did not occur even at a substrate concentration of 60 mM, and SAM was produced at approximately half the amount of the substrate concentration.

[0070] Experimental Example 5: Comparison of Purity and Optical Isomer Content of SAM (Mixture of SS-SAM and RS-SAM) Obtained by a Production Method According to an Embodiment of the Present Invention and Commercially Available SAM (Mixture of SS-SAM and RS-SAM) To examine the industrial usefulness of an embodiment of the present invention, HPLC analysis was performed using the method described above on a SAM obtained by a production method according to an embodiment of the present invention (a mixture of SS-SAM and RS-SAM; referred to as "the present invention") and a commercially available SAM (a mixture of SS-SAM and RS-SAM). Two types of commercially available SAM (referred to as "Comparative Example 1" and "Comparative Example 2"), both of which were commercially available from different companies, were prepared. The results of the above experiment are shown in Table 1. In Table 1, the "SAM" column indicates the sum of the HPLC percentages of SS-SAM and RS-SAM, and the "Other Impurities" column indicates the sum of the HPLC percentages of compounds other than SS-SAM and RS-SAM. As shown in Table 1, it was revealed that the SAM (a mixture of SS-SAM and RS-SAM) obtained by the production method according to the embodiment of the present invention had the highest purity.

[0071]

[0072] Furthermore, SAM can be divided into optical isomers, SS-SAM and RS-SAM, but only SS-SAM has physiological activity. In the cell extraction method, a typical method for producing SAM, racemization occurs during the production process, resulting in an RS-SAM content of 25-35%. On the other hand, enzymatically synthesized SAM is only SS-SAM. The optical isomer content ratios of SAM obtained by the enzymatic synthesis method of SAM according to an embodiment of the present invention (a mixture of SS-SAM and RS-SAM) and commercially available SAM (a mixture of SS-SAM and RS-SAM) were analyzed under the HPLC conditions described above. The results are shown in Table 2. As shown in Table 2, the SS-SAM content of each company was approximately 65 to 72%, whereas the SAM (a mixture of SS-SAM and RS-SAM) according to an embodiment of the present invention had an SS-SAM content of 95% or more, which was higher than that of other SAMs (mixtures of SS-SAM and RS-SAM) because it was enzymatically synthesized.

[0073]

[0074] Experimental Example 6: Advantages of SAM Obtained by a Manufacturing Method According to an Embodiment of the Present Invention as a Methyl Group Donor In this experimental example, the improvement in methyl group donating ability resulting from the high SS-SAM content of the SAM (a mixture of SS-SAM and RS-SAM) obtained by the manufacturing method of the present invention was verified.

[0075] The experiment in this example was carried out as follows. 50 μM substrate oligo RNA (5'pppGAA), 1x Capping buffer, 0.5 mM GTP, and 1 unit of Vaccinia capping enzyme (NEB, product number 2080) were mixed in a reaction solution, to which the SAM of Comparative Example 1 (a mixture of SS-SAM and RS-SAM) or the SAM of an embodiment of the present invention (a mixture of SS-SAM and RS-SAM) was added at a final concentration of 0.5 mM. The reaction was carried out at 37°C, and LC analysis was performed. The LC analysis conditions were as follows. A Kinetix EVO C18 column (particle size 1.6 μm, inner diameter 2.0 mm, length 150 mm) manufactured by Phenomenex was used. The mobile phase used was 20 mM potassium phosphate, 20 mM tetrabutylammonium, and 25% acetonitrile, with a flow rate of 0.2 mL / min and room temperature. For detection, 10 μL of the sample obtained by diluting the reaction solution 50-fold with the mobile phase was applied to the column, and the absorbance at 260 nm was measured to compare the amount of Cap0 produced. Cap0 is Cap0-pppGAA (= GAA with 7-methylguanosine added to the 5' end). Capping efficiency can be compared based on the amount of Cap0 produced.

[0076] The results of the above experiment are shown in Figure 4. As shown in Figure 4, the capping efficiency when using the SAM (a mixture of SS-SAM and RS-SAM) obtained by the production method according to an embodiment of the present invention was better than when using commercially available SAM (a mixture of SS-SAM and RS-SAM). It is believed that the 20% or higher content of physiologically active SS-SAM worked synergistically to enhance the methyl group-donating activity of the capping enzyme.

[0077] Accordingly, the present invention can be embodied in the following ways. <1> A solution containing boric acids, S-adenosylmethionine synthase, ATP, methionine, and water. <2> The solution according to <1>, wherein the concentration of the boric acids is 0.01 to 0.20 mol / L per 1 U of the S-adenosylmethionine synthase. <3> The solution according to <1> or <2>, further containing urea. <4> The solution according to <3>, wherein the concentration of the urea is 1.0 to 8.0 mol / L per 1 U of the S-adenosylmethionine synthase. <5> A method for increasing the amount of S-adenosylmethionine produced, comprising adding boric acids to a solution containing S-adenosylmethionine synthase, ATP, methionine, and water. <6> A method for improving the activity of S-adenosylmethionine synthetase, comprising adding boric acids to a solution containing S-adenosylmethionine synthetase, ATP, methionine, and water. <7> The method according to <5> or <6>, comprising further adding urea to the solution. <8> A method for producing S-adenosylmethionine using S-adenosylmethionine synthetase, comprising the solution containing S-adenosylmethionine synthetase, ATP, methionine, water, and boric acids. <9> The method for producing S-adenosylmethionine according to <8>, further comprising the solution containing urea.

Claims

1. A solution containing boric acid, S-adenosylmethionine synthetase, ATP, methionine, and water.

2. The solution according to claim 1, wherein the concentration of the boric acid compound is 0.01 to 0.20 mol / L per 1 U of the S-adenosylmethionine synthase.

3. The solution according to claim 1 or 2, further comprising urea.

4. The solution according to claim 3, wherein the concentration of the urea is 1.0 to 8.0 mol / L per 1 U of the S-adenosylmethionine synthase.

5. A method for increasing the amount of S-adenosylmethionine produced, which comprises adding boric acid to a solution containing S-adenosylmethionine synthase, ATP, methionine and water.

6. A method for improving the activity of S-adenosylmethionine synthetase, which comprises adding boric acid to a solution containing S-adenosylmethionine synthetase, ATP, methionine and water.

7. The method of claim 5 or 6, further comprising adding urea to the solution.

8. A method for producing S-adenosylmethionine using S-adenosylmethionine synthetase, comprising the step of preparing a solution containing S-adenosylmethionine synthetase, ATP, methionine, water, and boric acid.

9. The method for producing S-adenosylmethionine according to claim 8, further comprising the addition of urea to the solution.

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