JP2547438C - - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing a nucleoside compound using a base exchange reaction with a microorganism or a processed product thereof. [Background Art] A method for producing a new nucleoside by exchanging the base portion of a nucleoside with another base using an enzyme is described in, for example, JP-A-56-102794. This publication discloses Serratia marcescens, a mesophilic bacterium of the genus Serratia.
Disclosed is a method for producing 5-methyluridine or thymidine by subjecting thymidine to uridine or 2'-deoxyuridine to base exchange in the presence of potassium phosphate using a crude enzyme solution prepared from the cells of Marcescens IFO 3052. Have been. [Problems to be Solved by the Invention] Since the bacteria used in the above-mentioned conventional method are normal-temperature bacteria, they are easily deactivated in a short time at high temperatures and cannot be reused. Generally, the higher the temperature, the faster the chemical reaction such as the base exchange reaction. In general, the higher the temperature, the higher the solubility of the reactants. Therefore, if a symbol or enzyme capable of performing a base exchange reaction stably for a long time even at a high temperature can be used, the base exchange reaction can be performed at a high substrate concentration, so that a product can be obtained at a high concentration and the reaction can be performed at a high concentration. Time will also be reduced. However, a method for producing a nucleoside compound by a base exchange reaction using such a symbol or enzyme has not been known. [Means for Solving the Problems] To solve the above problems, the present inventors have proposed a thermophilic bacterium of about 100 grown at 65 ° C.
0 strains were searched. As a result, they have found that a thermophilic bacterium belonging to the genus Bacillus is excellent in heat resistance and can carry out a base exchange reaction at a high reaction rate, thereby completing the present invention. That is, the present invention provides a thymine, a ribonucleoside selected from the group consisting of inosine, adenosine and guanosine, or 2′-deoxyinosine;
Deoxyribonucleoside selected from the group consisting of 2'-deoxyadenosine and 2'-deoxyguanosine is combined with a thermophilic thermophilic bacterium of the genus Bacillus or an bacterium thereof in an aqueous solution in the presence of phosphoric acid or phosphate. It is a method for producing a nucleoside compound, characterized by producing a nucleoside compound which is 5-methyluridine or thymidine by a base exchange reaction with a processed body. The Bacillus thermophilic bacterium is preferably Bacillus stearothermophilus (Ba
cillus stearothermophilus) JTS 859, and the above base exchange reaction is usually performed at a temperature of 40 ° C to 70 ° C. Hereinafter, the present invention will be described in more detail. The present invention provides a base exchange between thymine and a specific ribonucleoside or deoxyribonucleoside using live cells of a thermophilic bacterium belonging to the genus Bacillus or a processed product thereof,
This is a method for producing the corresponding nucleoside compound, namely 5-methyluridine or thymidine. Bacillus thermophilic bacterium As the strain belonging to Bacillus stearothermophilus used in the method of the present invention, various known strains that have already been preserved can be used. However, the most preferred strain to be used is Bacillus stearothermophilus JTS859, which has a high enzyme production. This strain has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, and its deposit number is Seikoken Bacteria No. 9666 (FERM P-9666). The bacteriological properties of this strain were determined using the Burgess Manual of Systematic
The results of the study based on the analysis, Volume 2, are shown below. 1. Morphology: Bacillus: 5.4-6.5 μm × 0.7-0.9 μm Elliptical sporulation: 2.0-2.3 μm × 1.1-1.2 μm One per cell, position 2. Cultural properties NB medium: cultured at 62 ° C for 2 days. On a plate: white, slightly yellow. Shiny, opaque, non-swelling. The shape of the colony is circular wavy. On slant: white, slightly yellow. Shiny, opaque, non-swelling. Growth is moderate. 3. Biochemical properties 1 Gram staining positive 2 Anaerobic culture Does not grow 3 Mobility exists Pericardium 4 Oxidase positive 5 Catalase positive 6 Liquefaction of gelatin Liquefaction ability 7 Litmus milk Coagulation 8 OF test Fermentation type 9 VP, MR Test negative 10 Generation of gas from glucose No generation 11 Production of acid from glucose Produced 12 Production of acid from arabinose No production 13 Manufacture of acid from mannitol Produced 14 Production of acid from xylose No production 15 Growth on Sabouraud's medium Slant grown Liquid grown 16 Growth under 0.001% lysozyme No growth 17 Growth under 0.02% azide No growth 18.7 Growth under NaCl No growth (Up to 2% 19 Hydrolysis of casein with resolution 20 Hydrolysis of starch Decomposition Yes 21 Dihydroxyacetone generation Not generated 22 Egg yoke test No growth 23 Utilization of citric acid negative 24 Indole production negative 25 Urease activity positive 26 Phenylalanine deamination negative 27 Arginine dehydrolase activity positive 28 Tyrosine degradation negative 29 Levan production negative 30 Nitrate reduction positive 31 Denitrification ability of sodium nitrate negative 32 Production of hydrogen sulfide positive 33 Utilization of inorganic nitrogen source Growing with NH ₄ grown with NO ₃ as the only nitrogen source Growing with NH ₄ as the only nitrogen source 34 GC content 47.3% 35 Growth temperature Optimum for growth at 40-71 ° C 60-68 ° C 36 pH range Optimum for growth at pH 5.7-8.5 Optimum pH 6.0-7.0 Is identified as Bacillus stearothermophilus. In culturing the above strain, a medium containing tryptone, peptone or casamino acid as a nitrogen source, glucose or sucrose as a carbon source, and further containing a yeast extract and sodium chloride is used. The initial pH of the medium is 6.0-7.
0 is preferred. The culture is performed by shaking culture at 60 to 65 ° C. and 180 to 220 rpm or by aeration and agitation culture. The harvest time is preferably mid to late in the logarithmic growth phase. In the case of shaking culture, it corresponds to 4 to 6 hours after the start of culture. At the end of the cultivation for more than 7 hours, the specific enzymatic activity of the cells is drastically reduced. The strains collected in this manner can be used as they are or after treatment with live cells (these are collectively referred to as live cells, etc.). The treated product includes a supernatant obtained by crushing viable cells by ultrasonication and then centrifuging, or a supernatant obtained by heat-treating the supernatant at 65 ° C. for 1 hour and then centrifuging. Further, the supernatant may be treated with acetone to remove unnecessary proteins such as proteases to obtain a more stable form. Incidentally, the half-life of the enzyme activity at 63 ° C. of the enzyme in the crude enzyme solution obtained by crushing the cells was 392 to 401 hours. This value indicates that the half-life of the enzyme activity at 60 ° C. of the enzyme obtained from the conventional Serratia marcescens IFO 3052 is much longer than that of 1 hour. Production of Nucleoside Compound According to the present invention, in order to produce a nucleoside compound which is 5-methyluridine or thymidine, thymine is subjected to a base exchange reaction with ribonucleoside or deoxyribonucleoside in an aqueous solution using the above living cells. This reaction is performed in the presence of phosphoric acid or phosphate. Potassium phosphate can be used as the phosphate. The ribonucleoside that undergoes base exchange with thymine is selected from the group consisting of inosine, adenosine and guanosine. The deoxynucleoside is selected from the group consisting of 2'-deoxyinosine, 2'-deoxyadenosine and 2'-deoxyguanosine. The aqueous reaction solution may contain thymine, a ribonucleoside that is a ribose donor or a deoxyribonucleoside that is a deoxyribose donor, and a phosphate, and the above viable cells and the like are added thereto. The concentration of thymine in the aqueous solution is 20 to 10
0 mM, ribonucleoside or deoxynucleoside concentration is 20-100 m
The concentrations of M and phosphate are preferably 5 to 100 mM, but the reaction occurs even if the concentrations of thymine and ribonucleoside or deoxyribonucleoside are higher. The enzymatic reaction performed by the living cells and the like is an equilibrium reaction, and the equilibrium constant is, for example, 0.25 at 63 ° C. when thymine and inosine are reacted to produce 5-methyluridine and hypoxanthine. The reaction equilibrium constant for the reaction of thymine with 2′-deoxyinosine to produce thymidine and hypoxanthine is 0 at 63 ° C.
. 27. Therefore, the production concentration of 5-methyluridine or thymidine is determined by the initial concentrations of thymine and ribonucleoside or deoxyribonucleoside in the reaction solution. The reaction in the present invention proceeds to a theoretical value based on the equilibrium constant. The pH of the reaction solution is usually 6.0 to 8.5, preferably 6.5 to 7.0. The reaction can be carried out at 40 to 70 ° C., but the higher the temperature, the faster the reaction speed, and it is preferable to carry out the reaction at a temperature of 60 to 70 ° C. EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. Example 1 (A) Tryptone 10 g / l, yeast extract 5 g / l, glucose 3 g / l
, Glycerol 18.9 g / l and sodium chloride 3 g / l 100 ml of a liquid medium (pH 7.0) was placed in a 500 ml Erlenmeyer flask and sterilized. Five platinum loops of Bacillus stearothermophilus JTS859 were inoculated at 65 ° C.
The cells were shake-cultured at 35% relative humidity and 210 rpm for 4 hours. At this time, the growth of the fungus was in the middle stage of logarithmic growth. The obtained culture was centrifuged (1000 G, 10 minutes) to collect bacteria, and 260 mg of wet bacteria were obtained. (B) Inosine 5.36 g / l, thymine 2.52 g / l, monopotassium phosphate 4
. 08 g / l, dipotassium phosphate 5.22 g / l, and glucose 2.0 g / l
In a 50 ml Erlenmeyer flask charged with 10 ml of an aqueous reaction solution (pH 7.0), 130 mg of the wet bacterium obtained in (A) above was reacted at 65 ° C. for 3 hours. As a result of qualitative and quantitative analysis of the produced 5-methyluridine by high performance liquid chromatography, it was found that 1.57 g / l (6.1 mM) of 5-methyluridine was produced. This concentration is the theoretical value 6.6 mM calculated from the equilibrium constant 0.25.
Of 92%. If the reaction is continued further, the reaction proceeds to the theoretical value. For high performance liquid chromatography analysis, use a Shimadzu LC-
6A was used, and Chemcosolve 5-ODS-H φ 4 × 300 was used for the column. Water: methanol = 95: 5 (V / V) was used as the eluent, and the flow rate was 0.8 m.
Analysis was performed at a column temperature of 38 ° C. at 1 / min. Ultraviolet 254 nm was used for detection. Retention times for hypoxanthine, thymine, inosine and 5-methyluridine were 6.2, 8.7, 10.4 and 12.2 minutes, respectively. Example 2 400 mg of a wet bacterium obtained by culturing in the same manner as in Example 1 (A) was suspended in 4 ml of a 20 mM potassium phosphate aqueous solution (pH 7.0), treated with ultrasonic waves for 2 minutes, and centrifuged (12000 G). , 10 minutes) to obtain a supernatant. Inosine 22.35 g / l, thymine 10.50 g / l, monopotassium phosphate 1.
A reaction aqueous solution (pH 7.0) consisting of 36 g / l and dipotassium phosphate 1.74 g / l
4) The above supernatant (4 ml) was added to 36 ml and reacted at 65 ° C. for 16 hours. The amount of produced 5-methyluridine was 6.45 g / l, and the reaction had reached a complete equilibrium state. After the reaction, the reaction solution was converted to a cation exchange resin Dowex 50WX (H ⁺ type) 100m
1 to obtain a flow-through fraction containing thymine and 5-methyluridine, but not containing hypoxanthine and inosine. After concentrating this fraction, 5-methyluridine was isolated using high performance liquid chromatography to obtain 177 mg. For fractionation by high-performance liquid chromatography, use a pump manufactured by Shimadzu L
C-6A was used, and Lichrosolve RP-18 φ8 × 250 was used for the column. Water: methanol = 97.5: 2.5 (V / V) was used as an eluent at a flow rate of 4 ml / min. Ultraviolet 254 nm was used for detection. Example 3 50 μl of the supernatant prepared in the same manner as in Example 2 was treated with inosine 5.365 g / l, thymine 2.522 g / l, monopotassium phosphate 1.36 g / l and dipotassium phosphate 1
. 950 g of 742 g / l of an aqueous reaction solution (pH 7.0)
, 50 ° C, 60 ° C and 70 ° C for 1 hour, 2 hours and 3 hours. The results of analyzing the produced 5-methyluridine by high performance liquid chromatography are shown in Table 1 below. From this result, it is understood that the reaction rate increases as the reaction temperature increases. Example 4 The supernatant obtained in the same manner as in Example 2 was heated at 63 ° C. for 1 hour, and then centrifuged (1
2000G, 10 minutes) to obtain a supernatant. This supernatant was subjected to the following acetone treatment to partially purify, thereby removing unnecessary proteins. That is, 0.8 volume of −10 ° C. acetone was added to 1 volume of the supernatant, and the mixture was stirred for 15 minutes under ice cooling. This was centrifuged (12000 G, 10 minutes) to obtain a supernatant. 0.6 volumes of acetone at −10 ° C. were further added to the supernatant, and the mixture was stirred for 15 minutes under ice cooling. This was centrifuged (1200 G, 10 minutes), and the resulting precipitate was
Was dissolved in 20 mM potassium phosphate solution to obtain an acetone-treated enzyme solution. The recovery of the enzyme after the heat treatment was 90.6%, and that after the acetone treatment was 90.50%.
4%. In addition, the specific activity of the enzyme per protein increased 2.7 times in total. 50 μl of the thus-treated acetone-treated enzyme solution and 150 μl of a 20 mM potassium phosphate solution (pH 7.0) were mixed with a reaction aqueous solution (pH 7) containing 25 mM thymine, 10 mM monopotassium phosphate, 10 mM dipotassium phosphate and 25 mM ribonucleoside shown in Table 2. 0.0) was added to 800 μl, and reacted at 60 ° C. for 30 minutes. The generated 5-methyluridine was qualitatively and quantitatively analyzed by high performance liquid chromatography. The results are also shown in Table 2. Example 5 One volume of the supernatant obtained in the same manner as in Example 2 was added with 1.3 mM of thymine and 1.3 m of inosine.
After adding 3 volumes of a solution (pH 7.0) consisting of M and potassium phosphate 20 mM, the mixture was heated at 63 ° C. 100 μl of the obtained heat-treated crude enzyme solution was added to thymine 25m
M, 25 mM inosine and 20 mM potassium phosphate (pH
7.0) Added to 400 μm and reacted for 60 minutes. Table 3 below shows the heat treatment time and the concentration of the produced 5-methyluridine. From these results, the following equation representing the relationship between the activity of the enzyme and the heat treatment was obtained. logY = −7.5 × 10 ⁻⁴ T + log1.62 γ = −0.980 In the above formula, T is a heat treatment time (hour), and Y is 5-methyl generated when the above reaction is carried out using a heat-treated enzyme solution. Uridine concentration, γ, is the correlation coefficient. From this equation, it was found that the half life of the enzyme activity was 401 hours, indicating that the heat resistance was high. Example 6 10 mg of a wet bacterium obtained in the same manner as in Example 1 (A) was suspended in 200 μl of a 20 mM potassium phosphate solution (pH 7.0), and this was suspended in 25 mM of 2′-deoxyinosine.
A reaction aqueous solution consisting of 25 mM thymine and 20 mM potassium phosphate (pH 7.0)
) And reacted at 60 ° C. for 2 hours. As a result of qualitative and quantitative analysis of the generated thymidine by high performance liquid chromatography, 4
. It was found that 62 mM thymidine had been produced. This concentration has an equilibrium constant of 0
. This corresponds to 68% of the theoretical value of 6.83 mM calculated from 27. If the reaction is continued further, the reaction proceeds to the theoretical value. The high performance liquid chromatography analysis was performed under the same conditions as in Example 1. Retention times for hypoxanthine, thymine, 2'-deoxyinosine and thymidine were 6.2, 8.7, 12.7 and 18.3 minutes, respectively. Example 7 400 mg of a wet bacterium obtained by culturing in the same manner as in Example 1 (A) was suspended in 4 ml of a 20 mM potassium phosphate aqueous solution (pH 7.0), treated with ultrasonic waves for 2 minutes, and centrifuged (12000 G). , 10 minutes) to obtain a supernatant. The above solution was added to 36 ml of an aqueous reaction solution (pH 7.0) composed of 21.0 g / l of 2'-deoxyinosine, 10.50 g / l of thymine, 1.36 g / l of monopotassium phosphate, and 1.74 g / l of dipotassium phosphate. 4 ml of pure water was added and the mixture was reacted at 65 ° C. for 16 hours. The produced thymidine was 6.21 g / l, and the reaction had reached a complete equilibrium state. After the reaction, the reaction solution was converted to a cation exchange resin Dowex 50WX (H ⁺ type) 100m
1 to obtain a flow-through fraction containing thymine and thymidine but not hypoxanthine and 2'-deoxyinosine. After concentration of this fraction, thymidine was isolated using high performance liquid chromatography to obtain 158 mg. The fractionation by high performance liquid chromatography was performed in exactly the same manner as in Example 2. Example 8 100 μl of the supernatant prepared in the same manner as in Example 7 and 100 μl of a 20 mM potassium phosphate aqueous solution (pH 7.0) were mixed with 2′-deoxyinosine 25 mM, thymine 25 m
M, and a reaction aqueous solution (pH 7.0) consisting of 20 mM potassium phosphate 800 μm
The reaction was carried out at 40 ° C., 50 ° C., 60 ° C., and 70 ° C. for 1 hour, 2 hours, and 3 hours. The results of analyzing the generated thymidine by high performance liquid chromatography are shown in Table 4 below. From this result, it is understood that the reaction rate increases as the reaction temperature increases. Example 9 The supernatant obtained in the same manner as in Example 7 was heated at 63 ° C. for 1 hour, and then centrifuged (12,000 G, 10 minutes) to obtain a supernatant. This supernatant was subjected to the following acetone treatment to partially purify, thereby removing unnecessary proteins. That is, 0.8 volume of −10 ° C. acetone was added to 1 volume of the supernatant, and the mixture was stirred for 15 minutes under ice cooling. This was centrifuged (12000 G, 10 minutes) to obtain a supernatant. 0.6 volumes of acetone at −10 ° C. were further added to the supernatant, and the mixture was stirred for 15 minutes under ice cooling. This was centrifuged (1200 G, 10 minutes), and the resulting precipitate was
Was dissolved in 20 mM potassium phosphate solution to obtain an acetone-treated enzyme solution. The recovery of the enzyme after the heat treatment was 90.6%, and that after the acetone treatment was 90.50%.
4%. In addition, the specific activity of the enzyme per protein increased 2.7 times in total. 50 μl of the acetone-treated enzyme solution thus obtained and 150 μl of a 20 mM potassium phosphate solution (pH 7.0) were combined with a reaction aqueous solution (pH 7) comprising 25 mM thymine, 10 mM monopotassium phosphate, 10 mM dipotassium phosphate and 25 mM deoxyribonucleoside shown in Table 5. 0.0) was added to 800 μl, and reacted at 60 ° C. for 30 minutes. The generated thymidine was qualitatively and quantitatively analyzed by high performance liquid chromatography. The results are also shown in Table 5. Example 10 To 1 volume of the supernatant obtained in the same manner as in Example 7, 3 volumes of a solution (pH 7.0) composed of 1.3 mM of thymine, 1.3 mM of 2'-deoxyinosine and 20 mM of potassium phosphate were added. , 63 ° C. 100 μl of the obtained heat-treated crude enzyme solution
With 25 mM thymine, 25 mM 2'-deoxyinosine and 20 potassium phosphate
The reaction solution was added to 400 μl of an aqueous reaction solution (pH 7.0) consisting of mM and reacted for 60 minutes. Table 6 below shows the heat treatment time and the concentration of the produced thymidine. From these results, the following equation representing the relationship between the activity of the enzyme and the heat treatment was obtained. logY = −0.67 × 10 ⁻⁴ T + log2.10 γ = −0.989 In the above formula, T is the heat treatment time (hour), and Y is the concentration of thymidine generated when the above reaction is performed using the heat-treated enzyme solution. (MM) and γ are correlation coefficients. From this equation, it was found that the half life of the enzyme activity was 392 hours, indicating that the heat resistance was high. Comparative Example Using a crude enzyme solution prepared from the cells of Serratia marcescens IFO 3052, uridine or deoxyuridine was reacted with thymine at 37 ° C. and 60 ° C. in an aqueous solution in the presence of potassium phosphate, respectively. . The rate of formation of the resulting 5-methyluridine or thymidine was 2.8 times faster at 60 ° C than at 37 ° C. Next, when a reaction similar to the above was carried out using the crude enzyme solution which was previously heat-treated at 60 ° C. for 3 hours, 5-methyluridine or 37 ° C. was obtained at any of the reaction temperatures of 37 ° C. and 60 ° C. No thymidine was produced. From these results, it is possible to increase the production rate of 5-methyluridine or thymidine by increasing the reaction temperature. However, the enzyme prepared from the normal-temperature bacterium is inactivated at a high temperature and cannot be reused. It can be seen that the reaction cannot be performed over a period of time. [Effects of the Invention] As described above, in the present invention, the base exchange reaction between thymine and ribonucleosides or deoxyribonucleosides is carried out at a high temperature by using live cells of thermophilic bacteria of the genus Bacillus or processed cells thereof. As such, the corresponding nucleoside compound, ie, 5-methyluridine or thymidine, can be obtained at a high rate and at a high temperature. In addition, since the base exchange reaction can be performed at a high temperature, the problem of contamination by room temperature bacteria that cannot survive at a high temperature can be avoided. Furthermore, the bacterial cells used or the processed product thereof have an extremely long half-life of activity, and can withstand repeated long-term use.

Claims (1)

Claims (1) A group consisting of thymine and a ribonucleoside selected from the group consisting of inosine, adenosine and guanosine, or a group consisting of 2'-deoxyinosine, 2'-deoxyadenosine and 2'-deoxyguanosine A deoxyribonucleoside selected from among the following is subjected to a base exchange reaction with a live thermophilic bacterium of the genus Bacillus or a treated product thereof in an aqueous solution in the presence of phosphoric acid or phosphate to give 5-methyluridine or A method for producing a nucleoside compound, which comprises producing a nucleoside compound which is thymidine. (2) Bacillus thermophilic bacterium is Bacillus stearothermophilus JTS 859
The manufacturing method according to claim 1, wherein (3) The method according to claim 1 or 2, wherein the base exchange reaction is performed at a temperature in the range of 40 ° C to 70 ° C.