JPS626692A - Production of dicarboxylic acid - Google Patents

Abstract

PURPOSE:To obtain a dicarboxylic acid, efficiently by bringing a specific microorganism into contact with a straight-chain alkane and/or fatty acid ester. CONSTITUTION:A microorganism, consisting of Bacillus sp. TY-007 (FERM-P No.8219) obtained by separation from soil in upland field in OKINAWA KEN, belonging to the genus Bacillus and having the ability to produce a dicarboxylic acid by assimilating a straight-chain alkane and a fatty acid ester is inoculated into a culture medium containing the straight-chain alkane or fatty acid ester as a main carbon source and consisting of Na2HPO4, KH2PO4, MgSO4, FeSO4, yeast extract, etc., and culitvated at 40-70 deg.C under aerobic conditions for 2-10 days or microbial cells of the above-mentioned microorganism are added to a solution containing the straight-chain alkane and/or fatty acid ester and reacted therewith at 4-9pH and 40-70 deg.C for 1-10 days to afford the aimed dicarboxylic acid or dicarboxylic acid and monocarboxylic acid, hydroxycarboxylic acid, etc., corresponding thereto.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention utilizes microorganisms to produce dicarboxylic acids, particularly those having 4 carbon atoms.
The present invention relates to a method for producing ~18 dicarboxylic acids. Long-chain linear dicarboxylic acids obtainable according to the invention, such as adipic acid, pimelic acid.

Sebacic acid and the like are useful as raw materials for various chemical industries.

[Prior Art and Problems to be Solved by the Invention] Long-chain linear fatty acids have a wide range of uses as raw materials for surfactants, oil agents, stabilizers, and the like. However, these are beef tallow,
Since natural fats and oils such as coconut oil are used as raw materials, the production cost also fluctuates as the price of the raw material fats and oils fluctuates. Therefore, it is desired to establish an industrial production method that can supply these fatty acids at a stable price.

In addition, fatty acids obtained from natural fats and oils are limited to those with an even number of carbon atoms, but fatty acids with an odd number of carbon atoms exhibit specific properties in terms of melting point, surface activity, etc., making it possible to use these fatty acids economically. It is strongly desired that it be obtained.

Due to the above circumstances, a method for producing fatty acids using microorganisms is attracting attention. Regarding the production of dicarboxylic acids from hydrocarbons using known microorganisms,
Method using Pichia polymorpha (Special Publication Publication No. 45-24
392), a method using yeast of the genus Candida (Japanese Patent Publication No. 57-29994), and a method using yeast of the genus Torulopsis (Japanese Patent Publication No. 49-25186).

However, these conventional methods are not necessarily fully satisfactory in terms of the amount of dicarboxylic acid produced, and it is desired to establish a method with higher productivity.

[Means for solving problems]

The present inventor screened the natural world to search for new dicarboxylic acid-producing bacteria, and discovered that strains belonging to the genus Bacillus produce dicarboxylic acids by assimilating linear alkanes and fatty acid esters. I have reached the point where I have completed the .

That is, the present invention relates to a dicarboxylic acid ester, which is characterized by contacting and reacting a microorganism belonging to the genus Bacillus and having the ability to assimilate linear alkanes and fatty acid esters to produce dicarboxylic acids, with linear alkanes and/or fatty acid esters. A method for producing acid is provided.

Examples of microorganisms that belong to the genus Bacillus and have the ability to assimilate linear alkanes and fatty acid esters to produce dicarboxylic acids include Bacillus sp.
lus sp,) TY-007 (hereinafter referred to as TY-00
It is abbreviated as 7 bacteria. ).

The TY-007 bacterium was isolated by the present inventor from Okinawan field soil using the method described below.

The Sl medium components shown in the table below (excluding decane) were dissolved in distilled water 11, and the pH was adjusted to 7.

5 Qml of this medium was dispensed into a 500 mA' shaking flask and autoclaved at 120°C for 15 minutes. After cooling, 10 ml of decane was added aseptically. Add 1 liter of sterile water to this medium.
0mj! 1 g of soil suspended in l m I! The cells were cultured aerobically at 52° C. for 7 days without any additives.

Using the obtained culture solution, one platinum loop was streaked on a plate medium prepared by adding 20 g of agar to the above Sl medium. 52
The present invention was obtained by culturing at °C for 7 days and isolating the resulting colonies.

Danko Anno NaJPOa・12HzO1,5g KHzPO40
,5gMg504・7)1zo 0.2g Fe
50.・4Ht0 0.005g yeast extract 3
g N84NO33g Decane lQmf TY-007 Bacteria classification and identification method [Takeharu Haseyo.

[Classification and Identification of Microorganisms], Tokyo Publishing, 1976; Bergey's Manual of Determinant Hacteriology
Determinative Bacteriolo
gy), 8th edition, 1975].

Table 1 shows the results of the identification test for this Φ bacterium. In addition, the S bacteria Bacillus in the table
stearothermophilus (B, stearother
mo-philus), and 0 bacteria indicates Bacillus coagulans (B. coagulans).

5-1, 1, 1, 1, 1, 1, 2, 1, 2, 1, 2, 1, 2, 3, 3
Jumeki
Tengram stainability +2, Physiological properties Starch hydrolysis Ten catalase reaction Attitude towards oxygen
Facultative anaerobic salt tolerance (7%) - mutual bacteria 2 9 bacteria? Bacillus Bacillus No description No description + + + + d, v*t + ++ d+ No description No description 1 TY-007 bacterial phenotylalanine deamination reaction - Acid production ability from xylose medium Ten mannitol 〃
+Trehalose 〃
+ Glucose 〃 + Growth in anaerobic medium Ten-indole production ability
- Reduction of nitrates
Decomposition of casein
10 bacteria 2 1 bacteria 2 d d d d No description No description No description No description −1−1−+ d d d d Minative bacteriology, TY-007 bacteria is Durham staining, bacilli, catalase reactivity, It is classified as a member of the genus Bacillus based on the properties of its sprout fertilizer. In addition, based on its growth temperature range and properties such as milk fermentability, it is predicted that this bacterium is close to either Bacillus stearothermophilus or Bacillus coagulans, and its growth on Sabouraud dextrose agar medium, It was estimated that it was the closest bacterial species to Bacillus coagulans based on its growth in a medium containing 0.02% sodium azide at 55°C and its growth in an anaerobic medium. However, since they were different in other properties, the present inventor determined that it was a new bacterial species and named this strain Bacillus sp. ry-007. This page has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-8219.

In addition to the above-mentioned TY-007 bacterium, microorganisms of the genus Bacillus, which are mutant strains thereof and have the ability to assimilate linear alkanes and fatty acid esters to produce dicarboxylic acids, can be used in the present invention.

Hereinafter, the present invention using TY-007 bacteria will be explained.

The TY-007 bacterium is thermophilic and has the ability to assimilate linear alkanes and fatty acid esters to produce dicarboxylic acids.

To produce dicarboxylic acid using TY-007 bacteria,
This surface may be brought into contact with linear alkanes and/or fatty acid esters, for example, in a medium containing linear alkanes and/or fatty acid esters as the main carbon source.
Inoculate Y-007 bacteria and heat at 40 to 70°C, preferably 50°C.
Cultivation is carried out under aerobic conditions at ~65°C for 2-10 days, preferably 4-77 days. Here, as the linear alkane, those having 4 to 18 carbon atoms, particularly those having 6 to 16 carbon atoms are suitable. Specifically, n-butane, n-hexane, n-octane, n-decane, n-undecane, n-
-dodecane, n-hexadecane, etc. In addition, the fatty acid ester has 4 to 18 carbon atoms, especially 6 to 18 carbon atoms.
16 are preferred, specifically methyl butyrate and ethyl hexanoate.

Examples include ethyl undecanoate, methyl tetradecanoate, methyl hexadecanoate, and ethyl hexadecanoate.

In another embodiment of the contact reaction, the grown cells of the TY-007 bacterium obtained by culturing as described above are obtained by an operation such as centrifugation, and this cell is transformed into a cell containing the linear alkane and/or fatty acid ester. There is a method of adding it to a solution and allowing it to react. In this case, reaction conditions include a temperature of 40 to 70°C, preferably 50 to 65°C for 1 to 10 days, preferably 2 to 7 days.
A reaction of 1 day is appropriate. Moreover, pH 4-9 is suitable. In addition, a dicarboxylic acid can also be produced using a product that has been immobilized by a conventional method.

According to the method of the present invention, dicarboxylic acids having mainly 4 to 18 carbon atoms, such as glutaric acid, adipic acid, pimelic acid, speric acid (1,6-hexanedicarboxylic acid),
Sebacic acid (1,8-octanedicarboxylic acid), 1.9
-nonanedicarboxylic acid.

1.10-decanedicarboxylic acid, 1.12-dodecanedicarboxylic acid, 1.14-tetradecanedicarboxylic acid, etc. can be obtained efficiently. Further, in addition to dicarboxylic acids, corresponding monocarboxylic acids, hydroxycarboxylic acids, etc. can also be produced. Note that the product was quantified using a gas chromatograph. - [Example] Next, the present invention will be explained in detail with reference to Examples.

Example I NaJPO<・12Hz01.5g, KIIZPO
40.5g, NH4NO33g.

Mg5Oa ・7Hzo O,2g+ FeSO4・4
HzOO,005g and yeast extract 3g in distilled water 11
and adjusted to pH 7. 500ml of this medium!
The mixture was dispensed into 50 m6 shake flasks and autoclaved at 120°C for 15 minutes. After cooling, 1 ml of ethyl undecylate was added aseptically. Next, TY-007 bacteria (F
ERM P-8219) was inoculated and cultured aerobically at 52°C for 4 days.

After the culture was completed, the culture supernatant obtained by separating the bacterial cells was extracted with ether. The obtained extract was analyzed by gas chromatography. The results are shown in Table 1.

Example 2 Naz11PO4・1211z01.5g, KHzP
Oa O,5g, MgSO4'711200.5g,
Fe50.・41 (zo 0.013g, NHJ
Oi:h and 3 g of yeast extract were dissolved in distilled water 11 and adjusted to pH 7. 5 Qmji of this medium was dispensed into 500 ml shaking flasks and autoclaved at 120° C. for 15 minutes. After cooling, add 5 ml of n-undecane aseptically.
Added. Next, TY-007 bacteria (FERM
P-8219) was inoculated and cultured aerobically at 52°C for 4 days.

After the culture was completed, the culture supernatant obtained by separating the bacterial cells was extracted with ether. The obtained extract was analyzed by gas chromatography. The results are shown in Table 2.

Example 3 The same procedure as in Example 1 was carried out except that 0.5 m It of ethyl hexanoate was used instead of 1 ml of ethyl undecylate. As a result of analysis by gas chromatography, it was confirmed that 5.1 .mu./.beta. of 1.4-butanedicarboxylic acid was produced.

Example 4 The same procedure as in Example 1 was carried out except that 0.5 ml of methyl tetradecanoate was used instead of 1 m It of ethyl undecylate. The results are shown in Table 3.

Example 5 The same procedure as in Example 1 was carried out except that 0.5 ml of methyl hexadecanoate was used instead of 1 ml of ethyl undecylate. The results are shown in Table 4.

Example 6 The same procedure as in Example 1 was conducted except that 0.5 ml of ethyl hexadecanoate was used instead of 1 ml of ethyl undecanoate. The results are shown in Table 5.

Example 7 The same procedure as in Example 2 was carried out except that the culture temperature was changed from 52°C to 60°C. Gas chromatograph analysis results: 1,9 to nonanedicarboxylic acid 44.7
The production of ■/l was confirmed.

Example 8 The same procedure as in Example 2 was carried out except that 0.5 m It of n-hexane was used instead of n-undecane.

As a result of analysis by gas chromatography, 1.4-butanedicarboxylic acid 1 mg/j2. The production of 3 .mu./l of hexanoic acid was confirmed.

Example 9 The same procedure as in Example 2 was conducted except that 0.5 ml of n-hexadecane was used instead of n-undecane and the culture temperature was changed to 60°C.

The results are shown in Table 6.

〔Effect of the invention〕

According to the present invention, linear dicarboxylic acids useful as raw materials for chemical industries, such as adipic acid, pimelic acid, and sebacic acid, can be efficiently produced using microorganisms belonging to the genus Bacillus.

Official seal of the procedure chief) August 6, 1985

Claims (4)

[Claims] (1) A method for producing dicarboxylic acids characterized by contacting and reacting microorganisms belonging to the genus Bacillus and having the ability to assimilate linear alkanes and fatty acid esters to produce dicarboxylic acids with linear alkanes and/or fatty acid esters. Production method. (2) The method according to claim 1, wherein a microorganism capable of producing dicarboxylic acid is cultured in a medium containing a linear alkane and/or a fatty acid ester. (3) The method according to claim 1, wherein the reaction is carried out by adding a microorganism capable of producing dicarboxylic acid to a solution containing a linear alkane and/or fatty acid ester. (4) Microorganisms capable of producing dicarboxylic acids are Bacillus and
The method according to any one of claims 1 to 3, which is SP TY-007 (FERM P-8219).