JPH0139758B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for producing uracil using a microorganism belonging to the genus Clostridium. Uracil is useful as a pharmaceutical raw material or biochemical reagent. (Prior Art) Uracil is chemically synthesized by condensing formyl acetic acid and urea. Conventionally, the secretion of uracil-based compounds together with other nucleic acid components into the culture solution of microorganisms has been reported, for example, in Breuibacterium liquefaciens (The Journal of Bacteriology).
of Bacteriolgy (vol. 84, p. 1, 1962), Bacillus (Agricultural Biological Chemistry),
(Biological Chemistry), vol. 26, p. 586, 1962)
or other microorganisms (Japanese Patent Publication No. 40-10957, 52-139786, 53-38691). An example of a microorganism producing only uracil is a bacterium of the genus Brevibacterium (Tokukosho).
57-30476), bacteria of the genus Micromonospora (Special Publication No. 57-18873)
There is. However, it is not known that anaerobic bacteria accumulate uracil in the culture solution. (Problems to be Solved by the Invention) Although the above-mentioned methods are known, the chemical synthesis method for uracil has the drawback of requiring reaction under severe conditions. In addition, there are still many issues to be solved from a microbial perspective, and it is important to obtain bacteria that have high uracil production rates, high accumulation concentrations, or bacteria that accumulate only uracil in the culture solution and do not contain other ribonucleic acid components. It is. To this end, in addition to breeding and improvement based on known bacterial strains, the creation of new bacterial strains that produce uracil is an extremely important means. As a result of repeated studies based on these objectives, the present invention was achieved based on the discovery that microorganisms belonging to the genus Clostridium accumulate uracil in a culture medium. (Means for Solving the Problems) The present invention is a method for producing uracil, which is characterized by culturing a bacterial strain belonging to the genus Clostridium and having the ability to produce uracil, and collecting the produced and accumulated uracil. The microorganism used in the present invention belongs to the genus Clostridium and has the ability to produce uracil, and the microorganism described in the Examples of the present invention is the first such microorganism. The microorganisms used in the examples are considered to belong to the genus Clostridium in that they are obligate anaerobic spore-forming bacilli, but they are not well-known in terms of properties such as Gram stainability and ability to assimilate carbon sources, which will be described in detail later. It is different from bacteria of the same genus and is considered to be a new bacterial species. Since the official species name has not yet been assigned, this invention uses Clostridium sp.
Display No.S-1. Next, the production method and mycological properties of Clostridium sp. No. S-1 (hereinafter abbreviated as the present bacterium) will be described. (Creation method) This bacterium was isolated by the following method. That is, 5 ml of the liquid medium shown in Table 1 to which 1 g of sorbose was added was dispensed into test tubes, and after sterilization, a supply source was added in an anaerobic glove box, the tube was sealed with a butyl rubber stopper, and the gas phase was replaced with anaerobic gas. The cells were then cultured statically at 30°C, and subcultured approximately every 10 days. After sub-planting once in a liquid medium, add 30g of agar to the medium shown in Table 1.
, roll tube method using an agar medium supplemented with 1 g of sorbose (Methods in Microbiolcgy)
3B, p. 117 (1969)) was repeated to obtain this bacterium. (Mycological Properties) For examination of the mycological properties that indicate the mycological properties of the strain of the present invention, please refer to the “Anaerobe Laboratory Manual”.
Manual) 4th Edition” (The VIP Anaerobe
Laboratory Virginia Polytechnic Institute
and State University, Blacksburg (1972)) and Bergey's Manual of Determinative Bacteriology.
Manual of Determinative Bacteriology) No. 8
The method and culture medium composition described in "Classification and Identification of Microorganisms" (written by Takeharu Hasegawa, Gakkai Publishing Center) were used. (Microscopic findings) 1 Cell shape and size: Single or 3-4
Direct bacilli of Ren. Width: 0.4 to 0.8 μm, length: 3 to 8 μm 2 Flagella: having periflagella 3 Spores: present, terminal 4 Gram staining: negative (medium composition) Examples are shown in Table 1.

【table】

[Table] (Growth status) Growth on an agar medium containing the composition shown in Table 1 plus 30g/agar and 1g/sorbose is as follows. Shape: Circular Rim: Smooth Elevation: Slightly raised Surface: Smooth Color tone: White (physiological properties) Attitude towards oxygen: Obligately anaerobic Indole productivity: - Liquefaction of gelatin: - Catalase production Characteristics: - Starch hydrolysis: - Aesculin hydrolysis: + Pigment formation: - Sulfate reducing properties: - Milk: both coagulation and peptonization - Urease: - Vitamin requirement: None Growth range: Temperature 25~ 40℃, optimal temperature 35℃ PH
5.5 to 9.0, optimal PH7.0 (Characterization of carbon source) Add 5 ml of liquid medium containing the following carbon source (10 g/) to the basic medium shown in Table 1 to a test tube with a diameter of 18 mm to create a sterile medium. 80μ of this bacterium was inoculated, the gas phase was replaced with a sterilizing gas containing nitrogen (67%) and carbon dioxide (33%), and the culture was statically cultured at 30°C for 14 days. The growth is
Measure the turbidity at 600 nm using a spectrometer (Spectronic 20,
(manufactured by Shimadzu Corporation). If the turbidity at 600 nm differs from the control without carbon source by less than 0.1, it is "not assimilated," if it is 0.1 or more and less than 0.2, it is "slightly assimilated," and if it is 0.2 or more, it is "assimilated." And so. What is metabolized: raffinose, galactose, rhamnose, glycogen, trehalose, mannose, glucose, amygdalin, sorbose What is not metabolized: fructose, xylose, ribose, arabinose, maltose, sucrose, lactose, melibiose, cellobiose, melezitose, mannitol, starch ,
Adonitol, erythritol, inositol, methanol, ethanol, n-propanol, ethylene glycol, glycerol, formic acid, acetic acid, propionic acid, succinic acid (products from sugars, etc.) Assimilated into the basic medium shown in Table 1 in the above test 10 g of sugar was added, the gas phase was replaced with a sterilizing gas containing nitrogen (67%) and carbon dioxide (33%), and this bacteria was inoculated and cultured stationary at 30°C. Acetic acid and ethanol were produced as the main products in the medium using all carbon sources. (Comparison with conventional similar species) Based on the above mycological properties, this Clostridium sp. It is a bacterial strain. Moreover, it does not have the reducing property of sulfate. Based on these facts, this bacterium is considered to be a strain belonging to Clostridium. Gram-negative clostridia include Clostridium cellobioparum and Clostridium thermosatucaroliticum.
thermosaccharolyticum), Clostridium
Brevifuaciens (Clostridium)
brevifaciens), Clostridium kluyveri, Clostridium malacosomae, Clostridium thermocellum
thermocellum). Among these, Clostridium thermosatucaloriticum and Clostridium thermocellum can be distinguished from this bacterium in that they are thermophilic bacteria. Clostridium brevifuaciens and Clostridium malacosum can be distinguished from this bacterium in that they are alkaline bacteria. Clostridium cellobioparum only produces acetic acid and does not produce ethanol.
Clostridium kluyveri can be distinguished in that its only product is caproic acid. Based on the above, this strain is considered to be a new bacterial species belonging to the genus Clostridium, so it was named Clostridium sp. No. S-1. Furthermore, this strain was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as "FERM P-8852". (Cultivation method) The cultivation method is basically the same as that for general microorganisms, but it is necessary to prevent oxygen from entering, and in the laboratory, culture is carried out in an incubator tightly closed with a rubber stopper. , leaving it still or shaking it. On a slightly larger scale, a commonly used fermenter can be used as is, and an anaerobic atmosphere can be created by replacing the oxygen in the device with an inert gas such as nitrogen. The type of fermentation tank is not particularly limited, but in addition to commonly used stirring and mixing tanks, single-stage or multi-stage bubble column type, and draft tube type fermentation tanks can also be used. The carbon source used for culture may be any carbon source that can be assimilated, and the nitrogen source may be ammonium salts such as ammonium chloride or nitrates such as sodium nitrate.
Various nitrogen compounds that can be used in normal fermentation can be used. Other inorganic compounds such as potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, sodium chloride, iron sulfate, cobalt chloride, calcium chloride, zinc sulfate, copper sulfate, alum, sodium molybdate, boric acid, or biotin. Addition of vitamins such as yeast extract and yeast extract is generally carried out. This strain can also be used as a dormant bacterial cell in a method for producing uracil using carbon dioxide, which cannot be assimilated, as a raw material. In this case, the bacterial cells obtained by culture are separated from the culture solution by a commonly used method such as centrifugation or membrane filtration, and suspended in a culture solution in which the carbon source is removed from the normally used culture solution. Supply carbon dioxide gas instead of the source and cause the reaction to occur. Instead of carbon dioxide gas, dissolved carbon dioxide, carbonate, or hydrogen carbonate can also be added to the reaction solution. Unlike culture, uracil can be produced even in the presence of oxygen during the reaction. Uracil accumulated in the medium can be recovered by known techniques. The present invention will be explained below using specific examples. Example 1 Clostridium sp. No. S-1 strain was cultured as follows. 10g/
After adding 5 ml of sorbose to a test tube and sterilizing it, add 80μ of a culture solution in which the strain was pre-cultured in the same medium.
was added in an anaerobic glove box, and after sealing with a butyl rubber stopper, the gas phase was replaced with a sterilizing gas containing nitrogen (67%) and carbon dioxide (33%). at 30℃
When static culture was performed for 10 days, 1.5 mg of uracil was obtained in the culture solution. (Uracil was dispersed from the culture solution using high-speed liquid chromatography, and then confirmed to match the standard by mass spectrometry, ultraviolet absorption spectrum, and NMR spectrum.) Example 2 Culture was carried out in the same manner as in Example 1 by adding 10 g/amygdalin to the basal medium shown in Table 1. Static culture
2.5mg/uracil was produced in 14 days. Example 3 Culture was carried out in the same manner as in Example 1 by adding 10 g/glucose to the basal medium shown in Table 1. Static culture 14
1.9mg/uracil was produced per day. Example 4 Culture was carried out in the same manner as in Example 1 by adding 10 g/fructose to the basal medium shown in Table 1. Static culture
2.2 mg/uracil was produced in 14 days. Example 5 10 g/glucose was added to the basal medium shown in Table 1, and culture was carried out in the same manner as in Example. 1.1 mg/uracil was produced during 14 days of static culture. Example 6 Culture was carried out in the same manner as in Example 1 by adding 10 g/galactose to the basal medium shown in Table 1. Static culture
1.3 mg/uracil was produced in 14 days. Example 7 Culture was carried out in the same manner as in Example 1 by adding 10 g/trehalose to the basal medium shown in Table 1. Static culture
1.7 mg/uracil was produced in 14 days. Example 8 Culture was carried out in the same manner as in Example 1 by adding 10 g/raffinose to the basal medium shown in Table 1. Static culture
1.5mg/uracil was produced in 14 days. Example 9 Bacterial cells (dry weight 6 mg) were cultured in the medium shown in Table 1 using 10 g of sorbose as a carbon source.
was centrifuged, suspended in 5 ml of the medium shown in Table 1, and sealed with a butyl rubber stopper.
%) and carbon dioxide (33%), and reacted at 35°C for 4 days. 30mg/in the reaction solution
of uracil was produced. Example 10 In the same manner as in Example 9, bacterial cells (dry weight 1.6 mg) were suspended in 5 ml of the medium shown in Table 1, and after sealing with a butyl rubber stopper, the gas phase was replaced with nitrogen (33%) and carbon dioxide ( 33%) replaced with sterilizing gas containing air (33%),
The reaction was carried out at 35°C for 3 days. 5.2mg/in the reaction solution
of uracil was produced.

Claims (1)

[Claims] 1. A method for producing uracil, which comprises culturing a bacterial strain belonging to the genus Clostridium and having the ability to produce uracil, and recovering the produced and accumulated uracil.