JPH02207795A - Novel bacterium and production of 2-thiopheneacetic acid using the same bacterium - Google Patents

Abstract

PURPOSE:To efficiently produce 2-thiopheneacetic acid by using a bacterium belonging to the genus Streptomyces, capable of converting a 2-alkylthiophene into 2-thiopheneacetic acid. CONSTITUTION:An aqueous solution containing an inorganic salt such as phosphate and a nitrogen source such as urea is sterilized, blended with a 2- alkylthiophene such as 2-n-alkylthiophene and Streptomyces sp. KE-8891 bacterium (FERM P-10529) belonging to the genus streptomyces, capable of converting a 2-alkylthiophene into 2-thiopheneacetic acid, adjusted to pH6.0-8.0 and cultured aerobically by shaking culture or aerated spinner culture at 15-45 deg.C. Then the culture mixture is acidified with a mineral acid, insoluble substances are removed, the resulting substance is extracted with a solvent such as ethyl ether and the residue is recrystallized from water to give 2-thiopheneacetic acid. Consequently, 2-thiopheneacetic acid can be produced in high yield and high selectivity and further the substance having extremely high purity is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel microorganism and a method for producing 2-thiophene acetic acid using the same. More specifically, microorganisms that belong to the genus Streptomyces and have the ability to convert 2-alkylthiophene to 2-thiopheneacetic acid can be used as a chemical modifier for penicillin and cephalosporin by acting on 2-alkylthiophene. Certain 2-thiophene acetic acids (R,R, Chauvette et al., J.
Am, CheIII, Soc, +84+340
1 (1962)) with high yield and high selectivity.

[Prior art and problems to be solved by the invention] Generally 2
- Thiophene acetic acid can be produced by an organic synthesis method (1) chloromethylating thiophene, reacting with alkali cyanide to produce 2-cyanmethylthiophene, and hydrolyzing this (U.S. Pat. No. 2,533,084)
(2) A method of acetylating thiophene, further amidating it, and then hydrolyzing it (West German Patent No. 83275)
(3) A method in which thiophene is formylated and converted to 1-methylsulfinyl-1-methylthio-2-thienylethylene using formaldehyde dimethylcabutal-3-oxide (Japanese Unexamined Patent Publication No. 51-86458) , (4) A method in which thiophene is directly condensed with halogenated acetic acid by a photochemical reaction (Japanese Unexamined Patent Publication No. 53-46962), but all of them involve harmful reagents such as cyano compounds and expensive materials such as S-oxide. This method uses chemicals or high temperature and pressure.

In addition, the 2-thiophene acetic acid obtained by these methods is colored due to impurities caused by side reactions, etc., and the purification methods (JP-A-56-23429 and JP-A-6
1-76482) has been proposed.

[Means to solve the problem]

As mentioned above, in view of the fact that there are problems in producing 2-thiophene acetic acid using an organic synthesis method, the present inventors have conducted repeated studies to produce 2-thiophene acetic acid using microorganisms.

As a result, they discovered a microorganism that can oxidize 2-alkylthiophene to produce 2-thiopheneacetic acid, and completed the present invention.

That is, the present invention belongs to the genus Streptomyces, and 2-
A method for producing 2-thiophene acetic acid, characterized in that a microorganism capable of converting alkylthiophene into 2-thiophene acetic acid is allowed to act on 2-alkylthiophene, and a method for producing 2-thiophene acetic acid, which belongs to the genus Streptomyces, and converts 2-alkylthiophene into 2-thiophene acetic acid. Streptomyces sp. KB-8891 (FERM) has the ability to convert into acetic acid.
P-105λge).

The microorganism Streptomyces sp. KE-889.1 (hereinafter abbreviated as KE-8891) according to the present invention was isolated from soil near the Kanagawa Prefectural Industrial Research Institute using a medium such as mannitol yeast extract. It has been
This bacterium has the mycological properties shown below.

A. Morphology (1) Color tone of aerial hyphae: Gray-white (2) Condition of spore chain: Straight or bent (3) Production of melanin-like pigment: No production B. Growth condition in medium (1) Sucrose/nitrate agar The culture medium colony swells and spreads slightly to the surrounding area in a smooth manner. The surface is powdery and grayish white. The back side of the village is black-gray, with small black dots scattered within it. No production of water-soluble pigments was observed. It has a characteristic musty odor.

(2) Glucose-asparagine agar medium The colony is slightly raised in the center, the periphery is smooth, and the surface is grayish white at first, but gradually changes to light brown, brown, and reddish brown. However, some parts of the village remain grayish white. The underside of the colony is uniformly brown to reddish brown. No production of water-soluble pigments was observed. It has a characteristic odor (musty odor).

(3) Glycerin-asparagine agar culture medium Colonies are initially smooth and colorless, exhibiting a colony morphology similar to that of ordinary bacteria, such as Escherichia coli, but gradually the surface becomes rough and powdery, and the color changes from white to grayish white. Further, as the number of days of culture passes, the color becomes grayish white with a slight black tinge. The underside of the colony is dark brown. No production of water-soluble pigments was observed.

It has a characteristic odor (dusty odor).

(4) The starch inorganic agar medium colony is initially smooth and colorless, resembling a colony of ordinary bacteria (such as Escherichia coli), but later the surface becomes rough and the color becomes pale brown to pale pink. The surface of the village is pale pink to light brown;
The colonies are somewhat smaller and flatter than in other media. No production of water-soluble pigments.

Although there is no mold odor, it emits a characteristic odor.

(5) Tyrosine agar medium Growth characteristics are extremely similar to those on glycerin/asparagine agar medium. No melanin-like pigment production is observed.

(6) Skimmed milk medium Slightly blackish grayish-white bacterial cells were observed on the surface tube wall of the medium, coagulation of the milk was observed, and when cultured for a long time, digestion of the medium occurred slowly and the liquid became transparent.

The color of the medium remains unchanged.

C1 Physiological and biochemical properties (1) Growth temperature range: The optimum growth temperature is 25-35°C, and it can grow even at 45°C.

(2) Liquefaction of gelatin The Nigelcose peptone gelatin medium is liquefied.

(3) Starch does not undergo hydrolysis and bicomposition.

(4) Coagulation and peptonization of milk: Coagulation and peptonization in skim milk medium.
Peptonization (digestion) is recognized.

(5) Production of melanin-like pigment: Melanin-like pigment is not produced in either tyrosine agar medium or peptone yeast iron agar medium.

(6) Reduction of nitrate: No reduction was observed in broth containing 0.1% potassium nitrate.

(7) Production of hydrogen sulfide: No production was observed on peptone/yeast/iron agar medium.

(8) Catalase activity: Positive (9) Hemolytic (sheep): Negative θω Gram reaction: Negative (11) Free oxygen requirement: Obligate aerobic 02) Aesculin hydrolysis: Positive 0 ■ From urea Production of ammonia: positive 04 Production of indole: negative qω Production of acetyl methyl carbinol (VP test): negative 06) Utilization of citrate as a carbon source: negative ri,
Assimilation of carbon sources (on Pridham-Godelive agar medium) (1) L-arabinose: positive (growth) (2) D-xylose: positive (3) D-glucose: positive (4) D-fructose: positive (5) ) Sucrose: positive (6) Inositol: positive (7) L-rhamnose: positive (8) Raffinose: positive (9) D-mannitol: positive
determinativeBacteriolog
When searching based on y+ 8th edition, this bacterium was recognized as a new bacterium belonging to the genus Streptomyces. This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and its accession number is +7[! RM P-+os
2. 'l. The present invention includes all mutant strains that have the above-mentioned abilities, even if they are obtained by mutating the present bacterium naturally or by artificial means.

Next, a method for producing 2-thiophene acetic acid according to the present invention will be described.

The 2-alkylthiophene used in the present invention is usually
2-n-alkylthiophene represented by the following formula is preferred.

(In the formula, m is an integer from 2 to 12.) An aqueous solution containing an inorganic salt and a nitrogen source is sterilized, and 2-alkylthiophene and KE
Add -8891 bacteria and shake. After acidifying with mineral acid and removing undissolved substances by centrifugation, extraction is performed with a solvent such as ethyl ether. The desired 2-thiophene acetic acid can be obtained by recrystallizing the residue from water. The reaction temperature in this production method is set within the growth temperature range of the microorganism used, preferably within the optimum growth temperature range. Although it cannot be specified uniformly because it varies depending on the substrate, medium composition, pH, and other conditions, for example, 15 to 45°C
Preferably, the temperature can be set at 25 to 35°C. The pH of the reaction system is usually 6.0 to 8.0, preferably 6.8 to 8.0.
It is sufficient to set it within the range of 7.3.

Cultivation is usually carried out under aerobic conditions, for example, a shaking culture method or an aerated agitation culture method can be used. Culture time is 2-
The treatment may be carried out until thiophene acetic acid is sufficiently accumulated, usually for 4 days or more, preferably for 7 to 10 days. The substances added as inorganic salts include phosphates, magnesium salts, calcium salts, iron salts, and other trace metal salts as required.

The substance added as a nitrogen source may be any substance that can be assimilated by the bacteria used, and includes, for example, urea, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium phosphate, and various amino acids.

These nitrogen sources may be used at 11 fJ, or two or more types may be used in combination. Additionally, use vitamins as a nutritional source to promote bacterial growth.

An appropriate amount of yeast extract, malt extract, etc. may be added.

In the present invention, in addition to the case where the bacteria used are directly inoculated into a medium and cultured as described above, the bacteria used are also used as carbon sources that can be assimilated by the bacteria, such as glucose, fructose, sucrose, and maltose.

It also includes the case where bacterial cells obtained by culturing in advance in a culture solution containing molasses, starch, etc. are added to a solution containing a substance having the same composition as the above-mentioned medium, and the reaction is carried out.

〔Example] Hereinafter, the present invention will be specifically explained with reference to Examples.

Example 1 Medium 1 containing 0.25 g peptone + 25 g NaC1O + 0.25 g yeast extract and 0.5 g mannitol
Put 00 relatives into a 200 ml Sakaro flask and add 120
After heat sterilizing with ``C'' for 20 minutes, the pH was adjusted with NaOII.
was adjusted to 7.0. This was inoculated with two loopfuls of Streptomyces sp.
0 times/min) (preculture), then urea 0.2 g
, KHzPO40.2g, NaHzPOa o,
3g.

Medium 100 containing 08 g of MgSO4・7HzOo
Place the sample in a 200 d slope flask and heat at 120°C for 20 min.
After heat sterilization for a minute, the pH was adjusted to 7.0 with NaCl.

To this, 2-n-decylthiophene [formula (1), m = 1030.36 g] was added as the raw material 2-alkylthiophene.
After adding 200 μρ of the pre-cultured bacterial solution,
The cells were cultured at C for 7 days with reciprocal shaking (100 times/min).

The resulting culture solution was centrifuged, and the supernatant was made acidic with hydrochloric acid. 3 times with diethyl ether (50 mff1 each)
) Ether extraction was performed. A portion of the residue obtained by distilling off the ether from the ether solution was collected and converted into a methanol solution using a column with an inner diameter of 4.6 mm and a length of 250 mm filled with approximately 5 μm ODS (octadecyl silane) and a UV detector. Analysis was performed using a Hitachi L-6000 liquid chromatograph using methanol as an eluent at a flow rate of 1.0+1/min. The retention time of 2-thiopheneacetic acid was 2.67 minutes, and the retention time of the raw material 2-n-decylthiophene was 7.67 minutes.
Much shorter than 91 minutes (because the peaks were well separated,
Analysis was easy. As a result of analysis, it was found that 2-thiophene acetic acid was obtained with a yield of 95%.

In addition, as a result of measuring infrared absorption spectra and nuclear magnetic resonance spectra, the following data were obtained.
- Consistent with the data for thiophene acetic acid.

IR (niBr, cm-') 3050 (υ-e-N) 2900~2540 (-COOII) 1700 (υ
-c-o) 1398 (-CH2-) 1240 (-COO-) 1190, 1040. 850. 2-thiophene acetic acid was obtained with a yield of 688 (thiophene ring).

7.22 (m, ill, Is) Example 2~
6 As the raw material alkylthiophene in Example 1, 2
The reaction was carried out in the same manner as in Example 1 except that the following compounds were used in place of -n-decylthiophene, and the results were analyzed in the same manner as in Example 1, as shown in the table. Table 1 Streptomyces KE-8
Oxidation reaction of 2-n-alkylthiophene using 891 bacteria [Effects of the invention] The present invention is the first example of producing 2-thiophene acetic acid from 2-alkylthiophene using microorganisms.
- Thiopheneacetic acid can be stably produced with high yield and high selectivity. Furthermore, extremely pure substances can be obtained simply by recrystallizing from water. The 2-thiopheneacetic acid produced according to the present invention can be used as a chemical modifier for penicillin and cephalosporin.

Claims (2)

[Claims] (1) A method for producing 2-thiophene acetic acid, which comprises allowing a microorganism belonging to the genus Streptomyces and having the ability to convert 2-alkylthiophene to 2-thiophene acetic acid to act on 2-alkylthiophene. (2) Streptomyces sp. KE-8891 (FER) belongs to the genus Streptomyces and has the ability to convert 2-alkylthiophene to 2-thiophene acetic acid.
MP-10529).