JPS6387974A - Clostridium sp no.s-1 - Google Patents

Abstract

NEW MATERIAL:Clostridium sp No.S-1 which is a Gram-negative bacillus belonging to Clostridium genus and having peripheral flagellum. USE:Microbial strain for producing uracil, acetic acid and ethanol. PREPARATION:A liquid medium is added with sorbose, sterilized, added with microbial source in an anaerobic glove box, sealed and the gaseous phase is substituted with anaerobic gas. The strain is subjected to stationary culture at 30 deg.C and subcultured every 10 days.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application)
m) Concerning a new bacterium belonging to the genus. This bacterium can be used in methods for producing uracil and the like.

(Prior Art) Uracil is chemically synthesized by condensing formyl acetic acid and urea.

Conventionally, secretion of uracil-based compounds together with other nucleic acid components into the culture solution of microorganisms has been reported, for example, in Brevibacterium liquefaciens (Breuibacterium liquefaciens).
terit+m 1iqucfacicns) (
The Journal of Bacterium
anal of Bactcriolgy) Volume 84,
1, 1962), bacteria of the genus Bacillus (Agricultural Biological Chemistry)
l Chemistry), vol. 26, p. 586, 196
2) or other microorganisms (Special Publication No. 40-10957)
, 52-139786.53-38691). An example of a microorganism that only produces uracil is Brevibacterium.
) (Japanese Patent Publication No. 57-30476) and Micromonospora (HiCrO1llOnO3por) (Japanese Patent Publication No. 57-18873). However, although the above-mentioned methods are known, the chemical synthesis method of uracil has the drawback of requiring a reaction under the severe condition 1. In addition, there are still many microbial issues to be solved, such as the production rate of uracil,
It is important to obtain bacteria with a high accumulated concentration. To this end, in addition to breeding and improvement based on known bacterial strains, the creation of new bacterial strains that produce turacil is an extremely important means.

As a result of repeated studies based on these objectives, the present invention was achieved by successfully isolating a new anaerobic microorganism that produces uracil and the like.

(Means for Solving the Problems) The microorganism of the present invention is a Gram-negative bacillus that belongs to the genus Clostridium and has periflagella. Although it is considered to belong to the genus Clostridium in that it is an obligate anaerobic sporobacillus, it differs from known congener bacteria in various properties that will be described in detail later, such as Durham staining ability and ability to assimilate carbon sources. , is considered to be a new bacterial species. Since the official species name has not yet been assigned, this invention uses Clostridium sp. NQs.
-i is displayed. Next, Clostridium sp.
-1 (hereinafter abbreviated as the present bacterium) and its mycological properties are shown.

(Creation method) The native strain was isolated by the following method. That is, 5 m of the liquid medium shown in Table 1 with 1 g/l of sorbose added was dispensed into test tubes, sterilized, a supply source was added in an anaerobic glove box, the tube was sealed with a rubber stopper, and the gas phase was converted to anaerobic gas. The cells were replaced and cultured stationary at 30°C, and subculture was performed approximately every 10[ ]. After sub-planting once in a liquid medium, 30 g/1. The roll tube method (Methods in Microbiology) was performed using an agar medium to which 1 g/l of sorbose was added.
biology) Volume 3 B, p. 117 (1969)) was repeated 1 to obtain the home country.

・Manual (8 naerobe Laborator
y Manual) 4th edition J (The V, 1.P,
Anaerobe Laboratory Virgi
-nia Polytechnic In5titut
e and 5tate Universities, B
Bergey's Manual of Determinative Battalionology (1972) and Bergey's Manual of
i+etcrmtna-tive aacterto
+ogy) 8th Edition” “Classification and Identification of Microorganisms” (written by Takeharu Yoshiya, published by Gakkai Publishing Center),
The medium composition was used.

(Microscopic findings) 1. Cell shape and size 2. Single or 3-4 straight rods. Width 04~0.8μm, quality 3~8μτ~2. Flagella: have periflagellates, spores: present, terminal 46 Gram staining: negative (medium composition) Table 1 shows examples.

Table 1 (Growth status) The composition in Table 1 includes 30 g/l agar and 1 g/j! Growth on agar medium supplemented with sorbose is as follows.

Shape: Circular Circumference Edge: Round Smooth Elevation: Slightly raised surface Two circles Smooth Color Tone: White (Physiological properties) ■ Attitude towards oxygen: Obligate anaerobic ■ Productivity of indole ■ Liquefaction of gelatin ■ Productivity of catalase ■ Hydrolysis of starch ■ Hydrolysis of esculin: + ■ Formation of pigment ■ Reducibility of sulfide (assimilation of carbon sources) Adding the following carbon sources to the basic medium in Table 1 (Add 5 tdl of liquid liquid containing 109/j to a test tube with a diameter of 18 m, create a sterile medium, inoculate 80μ of the native culture, and change the gas phase to nitrogen (67%)
The cells were replaced with a bacteria-removal gas containing carbon dioxide (33%) and cultured stationary at 30°C for 14 days.

Growth was measured by measuring turbidity at 600 nm using a spectrometer (Spectronic 20, manufactured by Jiyu Seisakusho). If the turbidity of 600 rv differs from the control containing no carbon source by less than 0.1, it is "not assimilated", and if it is 0.1 or more and less than 0.2, it is "slightly assimilated". ``Capitalize'' the above
And so.

Assimilated: Roughinose, galactose.

Rhamnose, glycogen, trehalose, mannose, glucose, amyladalline. sorbostol, erythritol, inositol, methanol, ethanol,
n-propertool, ethylene glycol, glycerol, formic acid, acetic acid, propionic acid, succinic acid (products from sugars, etc.) Add 10 g/g of the sugars that were confirmed to be assimilated in the above test to the basic medium shown in Table 1. ffi was added, and the gas phase was nitrogen (67%
) and carbon dioxide (33%), and the native culture was inoculated and cultured 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. NQS-1 is an obligate anaerobic Durham-negative spore bacillus.
This is a strain whose main wJ fermentation metabolites are acetic acid and ethanol. It also has no ability to reduce sulfides. For this reason, in Japan, Clostridium (Clostridium)
It is thought that the strain belongs to M). Clostridium brevifaciens 11° (Clostridium brevifaciens
), Clostridium kluyveri (Clostridium
iui kluyveri) Clostridium malacosoma
e) Clostridium thermocellum (Clostri
diumthermocel ful). Among these, Clostridium thermosaccharum and Clostridium thermocellum can be distinguished from their home countries in that they are thermophilic bacteria. Clostridium brevifaciens and Clostridium malacosum can be distinguished from their home countries in that they are alkaline bacteria. Clostridium cellobiobalum can be distinguished in that it only produces acetic acid and does not produce ethanol, and Clostridium kluyveri can be distinguished in that it only produces caproic acid.

Based on the above, this strain is considered to be a new bacterial species belonging to the genus Clostridium, and therefore it was named Clostridium sp. NQS-1. Furthermore, this strain was approved by the Agency of Industrial Science and Technology (-Institute of Microbial Technology) as the
(FERM P-8852).

<Culture method) Cultivation method A is basically the same as for general microorganisms, but it is necessary to prevent oxygen from entering, and in the laboratory, it is used in an incubator tightly closed with a rubber stopper. ', a method of standing or shaking is used. On a slightly larger scale, a commonly used 111M tank 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 wJ fermenter is not particularly limited, but in addition to the commonly used stirring mixing tank, -stage or multi-stage bubble column type, and draft tube type fermenters 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 any of various nitrogen compounds that can be used in normal fermentation, such as ammonium salts such as ammonium chloride and nitrates such as sodium nitrate. .

Other as necessary, potassium dihydrogen phosphate, magnesium sulfate, manganese chloride, sodium chloride, iron sulfate, cobalt chloride, calcium chloride, zinc sulfate, copper sulfate, alum,
Inorganic compounds such as sodium molybdate and boric acid, or vitamins such as biotin and yeast extract are added as usual.

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, we will respond. 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.

(Effects of the Invention) By culturing the native culture, uracil, acetic acid, and ethanol can be accumulated in the culture medium. Uracil can also be accumulated in the medium by allowing carbon dioxide to act on microbial cells.

Uracil, acetic acid, and ethanol accumulated in the medium 1t-
It can be recovered using known techniques.

(Example of use) The present invention will be explained below using specific examples.

Usage Example 1 Clostridium sp. NfiS-1 strain was cultured as follows. 10 g/l of sorbose was added to the medium shown in Table 1 and dispensed into test tubes for 5 days. After sterilization, 80 μl of a culture solution in which the strain had been pre-cultured in the same medium was added in an anaerobic glove box, and the mixture was sealed with a butyl rubber stopper. Afterwards, the gas phase was replaced with a sterilizing gas containing nitrogen (67%) and carbon dioxide (33%).

When statically cultured at 30°C for 10 days, 1.5Rg/I of uracil was found in the culture solution! I got it. (After uracil was dispersed from the culture solution by high-speed liquid chromatography, it was confirmed that it matched the standard product by quality vIt analysis, ultraviolet absorption spectrum, and NMR spectrum).

Use Example 2 Culture was carried out in the same manner as in Use Example 1 by adding 109/ffi amygdalin to the basal medium shown in Table 1. 2.51 in 14 days of static culture! tg/l of uracil was produced.

Usage example 3 ICI/j! in the basic medium shown in Table 1! of glucose was added and cultured in the same manner as in Use Example 1. 1 in 14 days of static culture
．． 94I9/j! produced uracil.

Use Example 4 Culture was carried out in the same manner as in Use Example 1 by adding 10 g/l of fructose to the basal medium shown in Table 1. 2 in 14 days of static culture
．． 2Irg/j! of uracil was produced.

Use Example 5 Culture was carried out in the same manner as in Use Example 1 by adding 10 g/l glucose to the basal medium shown in Table 1. 1 after 14 days of static culture.
III! J/J! produced uracil.

Use Example 6 Culture was carried out in the same manner as in Use Example 1 by adding 10 g/l of galactose to the basal medium shown in Table 1. L after 14 days of static culture
3 ay/l of uracil was produced.

Usage example 7 10g/! in the basic medium shown in Table 1! of trehalose was added and cultured in the same manner as in Use Example 1. L after 14 days of static culture
7wi/l of uracil was produced.

Use Example 8 10 g/l of raffinose was added to the basal medium shown in Table 1 and cultured in the same manner as in Use Example 1. 1 in 14 days of static culture
．． Produced 5ay/jj of cattle.

Usage Example 9 Bacterial cells (dry seedlings 6Rg) cultured in the medium shown in Table 1 using 10 g/l sorbose as a charcoal source were centrifuged, suspended in 5IIl of the medium shown in Table 1, and treated with butyl rubber. After sealing with a stopper, the gas phase was replaced with nitrogen (67%) and carbon dioxide (
The mixture was replaced with a sterilizing gas containing 33%) and reacted at 35°C for 4 days. 30 IFJA:·1 nil of uracil was produced in the reaction solution.

Claims (1)

[Claims] Clostridium sp. No. 1 is a Gram-negative bacillus that belongs to the genus Clostridium and has periflagella. S-1