JPS602192A - Preparation of uracil form dihydrouracil - Google Patents

Abstract

PURPOSE:To prepare uracil efficiently, by treating a mold of Rhodotorula glutinis, a lyophilized mold, or an extract from the mold with dihydrouracil. CONSTITUTION:Rhodotorula glutinis IFO-0369 is capable of converting dihydrouracil to uracil in the mold even if it is cultivated in a medium using a common natural nutiritive source. When dihydrouracil is added to the medium, it further more converts dihydrouracil to uracil. So that, the mold of Rhodotorula glutinis, lyophilized mold, or an extract from the mold is reacted with dihydrouracil, to prepare uracil. By this process, about 0.4g/liter uracil can be accumulated in the reaction solution.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing uracil from dihydrouracil using a microorganism capable of converting dihydrouracil, /I/, to uracil.

An object of the present invention is to industrially advantageously produce uracil, which is widely useful in various fields such as medicine, agrochemicals, and chemistry.

Traditionally, uracil has been produced using chemical synthesis methods [Methods in Engineering 4, 626 (1957) published by Kyoritsu Shuppan Co., Ltd., Chemistry Dictionary Vol. 1, p. 791 (1967).
Journal of Applied Chemistry 2゜239 (1952)'; Helvetica Himika Acta 8,850 (1925)
] or fermentation method (mass culture of yeast etc.), or fermentation method (large scale cultivation of yeast etc.),
method of separating and collecting after extraction, hydrolysis, etc.) or fermentation accumulation method (Special Publication No. 49-22710, Special Publication No. 57-
18873, Japanese Patent Publication No. 57-30476, etc.).

However, chemical synthesis methods involve heating reactions under strong acids, or reactions under strong acids that require a reduction catalyst and a hydrogen stream. They have the disadvantage of requiring reactions under intense conditions.

In addition, in the fermentation method, it is necessary to extract the ribonucleic acid, hydrolyze it, and separate and collect the produced uracil using an ion exchange resin after cultivating yeast etc. in a large lid.
It has drawbacks such as a long incubation time, a complicated culture medium composition and a large number of them, and since the fermentation product contains substances other than uracil, it is necessary to separate and collect uracil using an ion exchange resin.

Therefore, unlike the conventional chemical synthesis methods, fermentation methods, and fermentation accumulation methods, which have many drawbacks as described above, a method for producing uracil in which the reaction proceeds under mild conditions and easily obtains uracil has been desired.The present invention As a result of intensive efforts to develop a method for producing uracil using enzymatic reactions using dihydrouracil as a raw material, they were able to react under much milder conditions compared to conventional chemical synthesis methods, fermentation methods, and fermentation accumulation methods. As a result of this progress, they discovered a method for producing uracil easily and completed the present invention.

The present invention relates to microbial cells of Rhodotorula having the ability to convert dihydrouracil to uracil, microbial cells subjected to freezing treatment, or extracts extracted from the microbial cells. relates to a method for converting dihydrouracil into uracil by acting on dihydrouracil.

The present invention also relates to a method for converting dihydrouracil into uracil by adding both phenanthroline and polyoxyethylene-alkylphenyl ether matha to the reaction solution in the above method.

In carrying out the present invention, microbial cells having the ability to convert dihydrouracil in the rhodotorula layer to uracil,
Alternatively, the frozen microorganism cells or the extract B extracted from the microorganism cells are added to an aqueous solution or an aqueous solution with an organic solvent (such as dimethyl sulfoxide in which dihydrouracil has good solubility) added to 06IW/V. %
Dihydrouracil dissolved at a concentration of preferably 0.2 w/v% to saturation yield is added to the seedling temperature or higher, preferably 20%
It is converted to uracil at ~45°C, preferably aerobically. Further, in the above method, during the reaction, phenanthroline, hapolyoxyethylene-alkylphenyl ether, or both are added to convert dihydrouracil to uracil.

In addition, uracil can be crystallized from the reaction mixture produced by the above-mentioned method by simply cooling the reaction solution after removing the used bacterial cells, or by concentrating and cooling, and uracil can be easily collected.

The present inventors believed that a method that utilizes an enzyme that allows the reaction to proceed efficiently under mild conditions is optimal for industrially converting dihydrouracil to uracil. The enzyme that converts dihydrouracil to uracil can be obtained by selecting from various sources such as animals, plants, and microorganisms, but it can be obtained cheaply and easily from microorganisms. is considered suitable. Useful microorganisms for this purpose are wild strains that exist in nature, strains stored in public microbial repositories, or strains that have been naturally or artificially mutated. The selection was made by examining the presence or absence of the ability to convert. As a method for testing this conversion ability, the present inventors used, for example, the following method. First) Cultivate the bacterial cells in a nutrient medium suitable for each strain.
Culture medium 11-100m/+ was collected by centrifugation and S buffered (p
After washing with H5-9), the obtained bacterial cells were divided into two equal parts and 24 x 20
Collect into a 0+++m test tube. One test tube contained 10 ml of buffer (pH 5-9) as a control, and the other tube contained 0.1-1.0 W/V% dihydrouracil-containing buffer (pH 5-9) as a reaction mixture. lQm/i addition 12
The reaction is maintained at 0 to 40°C and shaken, the converted uracil in the reaction solution is measured, and a strain having the ability to convert dihydrouracil to uracil is preselected.

In the above method, microorganisms capable of converting dihydrouracil to uracil were subjected to a shaking reaction at 30°C for 40 hours using a 0.5 w/v% dihydrouracil reaction solution (pH 7,4) to remove the uracil produced in the reaction solution. After measuring and searching, I found that Rhodotorula glutinis
a glutinis) engineering FO-0389 is 0.9P
/l, engineering FO-0415 is 0.21-/l.

FO-0688 (these are available from Fermentation Research Institute) was found to produce uracil in the reaction solution at 0.4 P/l, respectively, and among them, 1FO-0389 was superior in converting dihydrouracil to uracil. I saw that I had the ability to do that.

For the following examples, Rhodotorula glutinisae F.
The experiment was carried out using O-0389, but was not limited to just one strain, but was carried out using a strain belonging to the genus Rhodotorula that has the ability to convert dihydrouracil to uracil in the above-mentioned FO-0415 and FO-0688. Any of these can be applied, and it is also possible to induce a bacterial cell that has lost the ability to convert dihydrouracil to uracil by subjecting it to mutation treatment and use it in the present invention.

Rhodotorula glutinisae FO-038 used in the present invention
9. Even when cultured in a normal medium using general natural nutrient sources, the ability to convert dihydrouracil to uracil is produced and accumulated in the bacterial cells, or the medium contains dihydrouracil or uracil. It has been found that the ability to convert dihydrouracil to uracil can be further increased by this. The amount of dihydrouracil or uracil to be contained in the medium varies depending on the medium composition, economic efficiency, etc., but the amount used is usually selected from the range of 1 o, oiwyv% or more, preferably from 0.05 to 0.2 w/v%. or appropriate.

The culture conditions are a temperature of 15 to 35°C, preferably 20 to 30°C.
°CN 1 at pH 3-9, preferably pH 4-7
It is appropriate to culture for 5 to 50 hours. It was found that aeration and agitation during cultivation promoted an increase in the growth of microorganisms and an increase in the ability to convert dihydrouracil to uracil. In this way, as the culture progressed, the ability to convert dihydrouracil to uracil was generated and accumulated in the bacterial cells.

In addition, the present inventors have cultured and harvested Rhodotorula glutinisae PO-03891 cells for at least 1 hour, preferably for at least 12 hours, at -10°C or below, preferably at -2
It has also been found that freezing treatment at 0-70°C significantly increases the ability to convert dihydrouracil to uracil.

In addition, when performing a conversion reaction from dihydrouracil to uracil using 10-0389, we screened various additives to increase the reaction efficiency, and found that phenanthroline, a nonionic interfacial intercalator, and a sex agent. It has also been found that the conversion of dihydrouracil to uracil is dramatically increased by adding a certain polyoxyethylene-alkylphenyl ether or both to the reaction solution. As for its concentration, 1 phenanthroline is used in the reaction solution at a concentration of Q, lmM or more, preferably in the range of 0.5 to 5mM, and polyoxyalkylene-alkylphenyl ether is used in the reaction solution at a concentration of 0.05w.
/v% or more, preferably 0.05 to 0.2 W/
It is preferable to use it within the range of V%. As the phenanthroline that can be used, any one of "10-+-+P-7 enanthroline may be used, but 0-7 enanthroline is preferable. Also, as the polyoxyalkylene-alkylphenyl ether, available from Rohm & Eighth Co., Ltd. of the United States. Commercially available under the trade name Triton X-45,100,102
, 114, 165°305, and 405.

Since dihydrouracil is poorly soluble in water, method 1 of the present invention
During such a reaction, it is impossible to increase the substrate concentration above 1 w/v%. Therefore, using Rhodotorula glutinisae TO-0389 cultured in a nutrient medium containing dihydrouracil, dihydrouracil was added to a phosphate buffer (pH 7, 8) containing 0.35 w/v% dihydrouracil. 5 w/v% or more of dimethyl sulfoxide, which dissolves relatively well, preferably 10
~5. The reaction solution containing Ow/v% was incubated under aerobic conditions for 3
The reaction was carried out at 0° C. for 39 hours, and as a result, 77 to 89% of dihydrouracil was converted to uracil in the reaction solution to which 10 to 40 w/v% of dimethyl sulfoxide was added. Since no reaction inhibition was observed due to the addition of dimethyl sulfoxide, we found that by adding dimethyl sulfoxide to the number of reactions, it is possible to conduct the reaction with a higher concentration of dihydrouracil than in an aqueous solution reaction. O Rhodotorula glutinisae Fo-0389 can be easily collected by a simple centrifugation operation, for example, at 4000 rpm for 5 minutes, so it is convenient to use the book itself repeatedly, but in order to enable continuous reactions, It is advantageous to use immobilized bacterial cells, which has been done recently. The immobilization method is explained in detail in various reports and references ("Immobilized Enzymes" by Kazuki Chibata, published by J4 Dansha; "Enzyme Engineering" edited by Sanbe Fukui, Kazuchi Chibata, Shu Suzuki, Published by Tokyo Kagaku Doujin; etc.).

This bacterial cell was also immobilized using various comprehensive immobilization methods (polyacrylamide gel,
It is possible to immobilize the cells by enclosing them using polyvinyl alcohol, photocurable resin, polyurethane resin, konjac powder, gelatin, collagen, alginic acid, carrageenan, etc.). Rhodotrular Glutinisue FO-038 used in the present invention
When 9 bacterial cells were immobilized in calcium alginate gel and subjected to repeated shaking and aeration reactions, approximately 50% of the bacterial cells remained in the second reaction.
Compared to the case where the reactivity had decreased to 4〉, when immobilization was performed, the reactivity was maintained at more than 90% even in the second reaction, and by immobilization, the stability increased, We discovered the possibility of repeating the reaction of converting dihydrouracil to uracil.

According to the present invention Rhodotorula glutinisae FO-0389
When we investigated the reaction conditions for converting dihydrouracil into uracil, we found that the reaction hardly progressed under anaerobic conditions with nitrogen substitution, but the reaction progressed under standing, shaking, aeration, and stirring. It was found that the conversion reaction from dihydrouracil to uracil occurred more efficiently when the reaction was carried out under vaporization conditions.Therefore, the necessity of oxygen was found to increase the reaction efficiency, and the result was 0 or 10. After collecting the Dotorula glutinisae FO-0389 bacterial cells, dispersing them in a buffer solution, and then performing the usual cell disruption procedure (e.g., pressurized cell disruption using a French press, 11X press, Yedabress, etc., or ultrasonic treatment, Alternatively, the cell-free extract is treated with a ball mill, biprogen cell mill, dyno mill, etc.), and then centrifuged to remove the cell-free extract, which is then subjected to nucleic acid and ammonium sulfate fractionation. When reacting with dihydrouracil, we found that HtOz was released into the reaction solution at the same time as the conversion to uracil, although the details of the exact reaction mechanism are unclear.

In the production method of uracil by the fermentation method and fermentation storage ljf method,
It is necessary to separate and collect only uracil using an ion exchange resin, etc., but in the method for producing uracil according to the present invention, rhodotorula glutinisae FO-0,389i is allowed to act on dihydrouracil and converted to uracil. Thereafter, the reaction solution was sterilized by filtration, and crude crystals of uracil could be easily obtained by simply cooling the resulting clear reaction solution or concentrating and cooling.

Examples of the method of the present invention will be shown below, but the method of the present invention is not limited thereto.

In the examples shown below, the methods for quantifying uracil include separation and quantification by high performance liquid chromatography, and 2.
The increase in absorbance at 60 nm was measured by 26″On of uracil.
The same value was obtained with both the method of dividing by the molecular extinction coefficient of 8200 at m and the method of determining 0.

Example 1 Add 0.3w of Fumo extract to a 500-liter flask.
v%, malt extract Q, 3w/v%, polypeptone 0.5
Inject W/v%, glucose i, ow/v%, dihydrouracil 0.1 W/v% (pH 6,0) at 100 mA,
After autoclaving at 120°C for 15 minutes, three loops of Rhodotorula glutinisae FD-0389 were inoculated and
The culture was cultured with shaking at °C for 24 hours to prepare a preculture solution. Then 2
Same medium II as above in 1 volume mini jar! and Calalin (registered trade name: Sanyo Chemical Industries, Ltd., polyoxyalkylene glycol ether) 11, autoclaved at 120"C for 15 minutes, inoculated with 10 times of the above pre-culture solution, and heated at 27"C at 1600 rpm. 24 at vvm
Time aeration agitation culture was performed. After the cultivation was completed, the culture solution was collected by continuous centrifugation It (5000 rpm).
Lo, three 21s! A portion of the 0 wet bacterial cells obtained using a water mini-jar was stored in a freezer (approximately -30°C), thawed and thawed each time it was used, and added to a phosphate buffer solution (100°C).
After washing three times with mM pf17,4), it was subjected to a conversion reaction from dihydrorabucyl to uracil.\\.

Collect 0.53 P each of unfrozen bacterial cells and cryopreserved bacterial cells into a 24 x 200 mm test tube, and add phosphate buffer 71+ (10 QmM pH 7,8) containing 35 gudihydrouracil.
) was added for 10 minutes, and the shaking reaction was carried out at 30'C.
As shown in Table 1, the conversion rate of dihydrouracil to uracil by freezing at -30°C for 12 hours or more reduced the conversion rate of dihydrouracil to uracil to 11% of the unfrozen case. In contrast, more than 69% of dihydrouracil was converted to uracil.

Table 1 Example 2 Into a 500mA Yosaka Lof flask, add 03w of Fumo Extract
v%, malt extract 0.3 w/v%, polypeptone O,,5 w/v%, glucose 1. OW/v%,
Uracil 0. Pour 100m6 of IW/T% (pH 6,0), autoclave at 120°C for 15 minutes, inoculate 1000ml of FO-038,9 with three platinum loops, and culture with ruJ shaking at 30°C for 30 hours. After completing one culture, the culture solution was collected by centrifugation ltA (4000 rpm, 5 minutes), washed twice with phosphate buffer (10+nM pH 7,4), and dispersed in 3Q+7 with the same loose solution. Using the reaction solution shown in Table 2 to which 3 mΔ of the bacterial dispersion was added, 24
X 200 nn at 30°C in a test tube! The reaction was carried out, and the amount of uracil produced in the reaction solution after 20 hours was quantified as shown in Table 2. The conversion rate of uracil into uracil was 11% when no additive was added, whereas it showed a conversion rate of over 180%.

Table 2 Example 3 In the same manner as in Example 2, Rhodotorula glutinis 1F'0-0389 was cultured in a 500 mj Sakaro flask to obtain 491 wet bacterial cells. 1.2 y of the obtained wet bacterial cells were mixed with 2 y of water.
．． Dispersed at <8 m. After autoclaving at 120''C for 15 minutes and cooling to room temperature, 5W/V% alginium sodium chloride 4? was added to the above dispersion, and after mixing well, syringe 1 was used to inject Tris-containing lw/v% calcium chloride into the dispersion.
The above sodium alginate mixed solution was added dropwise into 500ml of hydrochloric acid buffer (10mM'pH 3, 4) with gentle stirring to produce bead-shaped immobilized bacterial cells with a diameter of about 3. After the dropwise addition, the mixture was left for another 1 hour at ≧1 temperature with gentle stirring. The obtained immobilized bacterial cells were collected by P paper filtration, and filtered with 50% solution containing 5mM calcium chloride on P paper.
After washing with mM Tris-HCl buffer, the fixed bacterial cells were incubated at 24X.
The sample was collected in a -200 zone test tube, the following reaction solution IQm6 was added, and the shaking reaction was repeated at 30°C. As a control for immobilization, 0.6 g of wet bacterial cells - 24X 20
The cells were collected in a 0-ball test tube, the following reaction solution IQ+a6 was added, and the shaking reaction was repeated in the same manner as for the immobilized cells.

As a result of quantifying uracil produced in the reaction solution, as shown in Table 3, the immobilized solid had higher stability during repeated reactions.

Table 3 Example 4 111i [
2, 12? After dispersing in 4.6 mt of water, 1 at 120°C.
5 w/v% sodium alginate solution f! autoclaved for 5 min. Using 1.8y, the actual MLi fAi
Fixation was performed in the same manner as in step 3, and the obtained fixed tooth body was divided into four equal parts, collected in a 21 x 200 rtm test tube, and mixed with the following reaction solution 1.
The temperature was maintained at 30'C, and the reaction was repeated in an aeration tI apparatus using a glass badger. As a control for immobilized bacterial cells, one frozen cell was incubated at 21x2oO
The cells were collected in a test tube, and the following reaction solution IQm8 was added, and the reaction was carried out in the same manner as for the immobilized cells. As shown in Table 4, the results of quantifying the amount of uracil produced in the reaction bath liquid showed that the stability during repeated reactions increased with each immobilization.

reaction solution 0 indicates conversion rate %.

Example 5 After thawing 8 g of the bacterial cell Rhodotorula glutinisae FC+ -0389 obtained in Example 1 and stored frozen at -30°C for 20 days, phosphate buffer (20 mM pH 7,
Wash 3 times with 8) and disperse in 20m6 of the same buffer for 15-20 minutes
The mixture was kept at 0.degree. C. and subjected to ultrasonication for about 30 minutes, followed by centrifugation (110,000 rpm, 10 minutes) to obtain a cell-free extract. The conversion reaction of dihydrouracil to uracil using this cell-free extract was carried out using a 24 x 200 m test tube at 30°C and a shaking reaction. The results are shown in Table 5.
0-phenanthroline and Triton X-10, such as
74-82% of dihydrouracil was converted to uradino-k in 19 hours with or without the addition of 0.

Example 6 After thawing the bacterial cell Rhodotorula glutinisae FO-0389 obtained in Example 1 and stored frozen at -30°C for 40 days,
Wet weight 49P and 100mM phosphate buffer (pH 7,8) containing 11 dihydrouracil 3.5y-, calalin 4y, and 1mM 0-phenanthroline 11 and 21
The mixture was placed in a mini-jar, maintained at 30°C, and subjected to an aeration-stirring reaction for 47 hours. After the reaction, centrifugation (4000r
pm, 5 minutes) to obtain a clear liquid of 975 mt.

When this clear liquid was cooled and stored in a refrigerator, needle-shaped crystals were obtained. Beat this crystal with hard F paper, wash with a small amount of ethanol on IP paper, dry at about 60℃,
56? crystals were obtained. When this crystal sample 1 was examined by high-performance liquid chromatography, it showed a purity of 97.4% (yield 45.4%).0 crystal sample I was redissolved in water and recrystallized. The original purple analysis of the refined @muku product obtained by repeated tests was O: 42.89%, H: 3.57%, N: 25.
00%, o; 28.54% (theoretical value 0-': 42.
86% +' H: s, 6o%, N: 24.99
%lO: 28.55%), and its UV spectrum matched well with that of pure uracil.

The mother liquor from which the previous crystals were removed was concentrated at about 970 mA in an evaporator at about 100 i#t, and then stored in a cold storage room to form crystals.The obtained crystals were beaten with hard P paper [after filtration, A crystal of 1.1.35F was obtained by washing on paper with a small amount of ethanol and drying at about 60°C. When this crystal specimen B was examined by high-performance liquid chromatography, it showed a purity of uracil of S52.8% (yield 51.4%) 0 Procedural amendment (voluntary) Mr. Kazuo Wakasugi, Commissioner of the Japan Patent Office, 1. Display 1982 Patent Application No. 109993 2
, Name of the invention: Process for producing uracil from dihydrouracil 3, Relationship with the case of the person making the amendment Patent applicant\Iryo Oboro N\Name: Nagase Seikagaku Kogyo Co., Ltd. 4, Agent 6, Contents of the amendment (1) From the bottom of page 11 of the specification, in line 4, "polyoxyalkylene" is corrected to "polyoxyethylene."

(2) On page 12, line 3, “polyoxyalkylene” is replaced with “
Polyoxyethylene” is corrected.

(3) On the last line of page 15, ``by p-filtration'' is corrected to ``by filtration or centrifugation, etc.''.

(4) On page 17, line 2, "polyoxyalkylene glycol ether" is corrected to "polyalkylene glycol ether."

(5) From the bottom of page 20 @ lines 5 to 4 “Wet bacterial cells 1.2
Water 2.8 ff1l! 'J is corrected to [0.69 wet bacterial cells is 1.4'ml'J of water.

(6) From the bottom of page 20, in the second line, "4 ml of sodium alginate" is corrected to "2 g of sodium alginate."

(Line 10 from the bottom of page 22 [wet weight 2.129 to 4.5 me J] [wet weight 1.069 to water 2.3
Correct me J.

(8) From the bottom of page 22, in line 8, "8g" next to "sodium alginate solution" is corrected to "4g."

(9) From the bottom of page 22, in line 6, "divide into four equal parts" is corrected to "divide into two equal parts."

that's all

Claims (1)

[Scope of Claims] 1. Any of microorganisms belonging to the genus Rhodotorula having the ability to convert dihydrouracil into uracil, microorganisms subjected to freezing treatment, or extracts extracted from the microorganisms. 1. A method for producing uracil from dihydrouracil, which comprises causing uracil to act on dihydrouracil. 2. Either a microorganism belonging to the genus Rhodotorula that has the ability to convert dihydrouracil into uracil, a frozen microorganism, or an extract extracted from the microorganism is allowed to act on dihydrouracil. A method for producing uracil from dihydrouracil, characterized in that 7-enanthroline, polyoxyethylene-alkylphenyl ether, or both are added to the reaction solution. 3. The method according to claim 1 or 2, wherein the microbial cells are immobilized microbial cells. 4'. The method according to claim 1 or 2, wherein the microbial cells are frozen and treated microbial cells or immobilized microbial cells. 5. The method according to claim 1 or 2, in which the freezing treatment is carried out at -10°C or lower for 1 hour or more. 6. The claim in which the freezing treatment is carried out at -20 to -70°C for 12 hours or more. The method described in Section 5. 3. The method according to claim 2, wherein the 7,7 enanthroline is 0-7 enanthroline. 8. The method according to claim 7, wherein Q, lmM or more of 8,o-7 enancetetraphosphorus is used. 9. The method according to claim 8, wherein 0.5 to 5 mM of 9, O-7 enanthroline is used. 10, Ho! 3. The method of claim 2, wherein the J oxyethylene-alkylphenyl ether is polyoxyethylene-pt-octylphenyl ether. 11. The method according to claim 10, wherein polyoxyethylene-pt-octylphenyl ether is used in an amount of 0.05 W/v% or more. 12. The method according to claim 11, wherein 0.05 to 0.2 w/v% of polyoxyethylene-pt-octylphenyl ether is used. 13. Microbial cells belonging to the Rhodotorula layer are FO203
89, IFO-0415 or IFO-0688.