JPS626672A - Bacillus sp. ty-007 and production of monocarboxylic acid using same - Google Patents

Abstract

PURPOSE:To efficiently produce a monocarboxylic acid useful as an intermediate for organic syntheses, a raw material for polymers, etc., by subjecting a specific bacterium and a straight-chain alkane and/or fatty acid ester to a contact reaction. CONSTITUTION:Thermostable Bacillus sp. TY-007 (FERM P-8219), separated from soil of plowed field in OKINAWA Prefecture, capable of assimilating a straight-chain alkane and/or fatty acid ester and producing a monocarboxylic acid, is inoculated into a medium containing 4-18C straight-chain alkane (e.g., n-butane) and/or 4-18C fatty ester (e.g., ethyl hexanoate), cultivated aerobically at 40-70 deg.C for 2-14 days, or the bacterium is added to a solution containing the straight-chain alkane and/or fatty acid ester and reacted at 4-9pH at 40-70 deg.C for 1-10 days to give 4-18C monocarboxylic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to Bacillus sp. TY-007, a monocarboxylic acid producing bacterium, and a method for producing monocarboxylic acids using the microorganism. The obtained monocarboxylic acid is useful as an intermediate for organic synthesis, a raw material for polymers, etc.

[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-mentioned circumstances, fatty acid production methods using microorganisms are attracting attention. Conventionally, as a method for producing monocarboxylic acids using microorganisms, a method for producing monocarboxylic acids from n-alkanes using Bacillus stearothermophilus is known. Furthermore, a method for simultaneously producing monocarboxylic acid and dicarboxylic acid from n-alkanes using yeast belonging to the genus Candida has also been proposed (Japanese Patent Publication No. 57-29994).

However, these are not fully satisfactory, and it is desired to establish microorganisms with better monocarboxylic acid production ability and an efficient method for producing monocarboxylic acids using such microorganisms.

[Means for solving problems]

The present invention is directed to Bacillus sp., which is thermophilic and has the ability to assimilate linear alkanes and fatty acid esters to produce monocarboxylic acids.
, ) TY-007 (hereinafter abbreviated as TY-007 bacteria) and a method for producing a monocarboxylic acid, which is characterized by contacting and reacting the microorganism with a linear alkane and/or fatty acid ester.

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

The sr medium components shown in the table below (excluding decane) were dissolved in distilled water 1β, and the pH was adjusted to 7.

50 ml of this medium was dispensed into a 500 ml shake flask and autoclaved at 120"C for 15 minutes. After cooling, 10 mJ of decane was added aseptically. To this medium, 1 ml of sterile water was added.
1 m/ml of 1 g of soil TFI suspended in 0 ml was added and cultured aerobically at 52°C for 7 days.

Using the obtained culture solution, a platinum loop was streaked onto a plate medium prepared by adding 20 g of agar to the above SI medium. 52
The bacteria were cultured at ℃ for 7 days and the resulting colonies were isolated to obtain the present bacteria.

sr medium NazllPO, 12HzO1,5g KHzPO
,0.5gMgSO4'711z0 0.2g F
e5Oa・4HzOO, 005g yeast extract 3
g N11tN0,3g Decane 10m6 TY-007 Bacterial classification and identification method [Takeharu Haseyo.

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

Table 1 shows the results of the identification test for this bacterium. In addition, Si in the table
Stealothermophilus (B, stea)
B. coagulans (B. coagulans).

1st appearance Bacillus flagellum Periflagellar motility
ten sprout fertilizer
Tengram stainability +2, Physiological properties Starch hydrolysis Tencatalase reaction Ten Attitude towards oxygen
Facultative anaerobic salt tolerance (7%) - Mutual bacterium 2 Bacillus No description No description + + Bacillus + d, v112 + + + d + No description No description Mycological alliance Enimitate 1M phenylalanine depletion Amino reaction - acid production ability from xylose medium 10 mannitol
+Trehalose 〃
+Glucose〃 +Growth in anaerobic medium Tenindole production ability -Nitrate reduction
Decomposition of casein
10 * 2 d: Difference in reaction V: Same strain, indeterminate bacteria, two M? d d d d No description No description No description No description -10-+ -+ d d d d Tig Bacteriology, -It is classified into the genus Bacillus based on its reactivity and spore properties. Furthermore, based on its growth temperature range and properties such as lactic acid fermentation, this bacterium is predicted to be a species close to either Bacillus stearothermophilus or Bacillus coagulans, and its growth on Sabouraud dextrose agar medium and Aji It was estimated that it was the closest bacterial species to Bacillus coagulans based on its growth in a medium containing 0.02% sodium 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 fungal species and named the woody fungus Bacillus sp. TY-007 strain. This bacterium was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-821.
It has been deposited as No. 9.

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

In order to produce monocarboxylic acids using the TY-OQ 7 bacteria, it is sufficient to contact the bacteria with linear alkanes and/or fatty acid esters, for example, using linear alkanes and/or fatty acids as the main carbon source. The TY-007 bacterium is inoculated into a medium containing Esther and cultured under aerobic conditions at 40 to 70°C, preferably 50 to 65°C, for 2 to 14 days, preferably 3 to 10 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-
Examples include undecane, n-dodecane, and n-hexadecane. Further, as the fatty acid ester, those having 4 to 18 carbon atoms, particularly those having 6 to 16 carbon atoms are preferable, and specific examples include methyl acetate, ethyl hexanoate, ethyl undecanoate, and methyl tetradecanoate.

Examples include 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. In addition, p) (4 to 9) are suitable, and monocarboxylic acids can also be produced by immobilizing the above bacterial cells by a conventional method.

According to the method of the present invention, monocarboxylic acids mainly having 4 to 18 carbon atoms, such as butyric acid, hexanoic acid, octanoic acid, nonanoic acid, undecylic acid, tetradecanoic acid, hexadecanoic acid, etc., can be efficiently obtained. Further, in addition to monocarboxylic acids, corresponding dicarboxylic 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 NazHPO, -1211z01.5g, KH2PO
40.5g, MgSO4'711□00.2g, F
eSO4・4H200,005g, Nil, No: 1
Dissolve 3 g of yeast extract and 3 g of yeast extract in distilled water,
Adjusted to H7. Transfer this medium to a 500 ml shake flask. It was dispensed and autoclaved at 120°C for 15 minutes. After cooling, 1 mg of ethyl undecylate was added aseptically. 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 1.

Example 2 Na2HPO4' 12Hz01.5g, KH2PO
40.5g, MgSO4・711200.5g, F
eSO4・4Hz00.013g, N114NO33
Dissolve g and 3 g of yeast extract in distilled water, pH 7.
Adjusted to. 500mj of this medium! 50 mJ was dispensed into a volume shaking flask and autoclaved at 120°C for 15 minutes. After cooling, 5 ml of n-undecane was added aseptically.

Next, TY-007 bacteria (FERM P-8
219) and cultured aerobically at 52°C for 4 days.

After the culture was completed, the culture supernatant obtained by separating the bacterial cells was subjected to E-chill extraction. 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 ml of methyl tetradecanoate was used instead of 1 mj2 of ethyl undecylate. The results are shown in Table 3.

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

Example 5 The same procedure as in Example 1 was carried out except that 0.5 ml of ethyl hexadecanoate was used instead of 1 m6 of ethyl undecanoate. The results are shown in Table 5.

Example 6 The same procedure as in Example 2 was conducted except that 0.5 m 12 of n-hexane was used instead of n-undecane and the culture was cultured at 52° C. for 3 days. As a result of analysis by gas chromatography, hexanoic acid 3/l and 1,4-
Production of 1 mg/l of butanedicarboxylic acid was confirmed.

Example 7 The same procedure as in Example 2 was conducted except that 0.5 mA 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, monocarboxylic acids useful as foods, organic synthesis intermediates, and polymer raw materials can be efficiently produced using a novel microorganism.

Procedural amendment stamp button August 6, 1985

Claims (4)

[Claims] (1) Bacillus sp. TY-007 is thermophilic and has the ability to assimilate linear alkanes and fatty acid esters to produce monocarboxylic acids. (2) Contact reaction of Bacillus sp. TY-007, which is thermophilic and has the ability to assimilate linear alkanes and fatty acid esters to produce monocarboxylic acids, with linear alkanes and/or fatty acid esters. A method for producing a monocarboxylic acid characterized by: (3) The method according to claim 2, wherein Bacillus sp. TY-007 is cultured in a medium containing a linear alkane and/or a fatty acid ester. (4) The method according to claim 2, wherein the reaction is carried out by adding grown cells of Bacillus sp. TY-007 to a solution containing a linear alkane and/or fatty acid ester.