JP3069965B2 - Method for producing dialcohol - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a process for the production of the corresponding dialcohols by microorganisms from fatty acids or their esters or salts, methyl ketone or monohydroxymethyl ketone, or mixtures thereof. Dialcohols are expected to be used as raw materials for synthetic high molecular substances that are polycondensed with dibasic acids such as adipic acid and terephthalic acid.

[0002]

2. Description of the Related Art
Dialcohols are only slightly produced by a chemical synthesis method, and no effective information has yet been obtained on their general properties and uses. The present dialcohol is one of the metabolites of fatty acids of microorganisms, and the production of methyl ketone and hydroxymethyl ketone, which are intermediate metabolites by microorganisms, has been proposed by the present inventors (Japanese Patent Application No. Sho 6).
3-320694, Japanese Patent Application No. 2-44386, etc.).

[0003]

Means for Solving the Problems The present inventors proceeded with detailed studies on the properties of microorganisms capable of assimilating fatty acids or esters thereof and converting them to the corresponding methyl ketone. The present inventors have found that some microorganisms belonging to the present invention produce dialcohols in high yield and completed the present invention. In other words, the general formula Formula _{1 R1-CH 2 -R2- (CH} 2) 2 -COO
H (wherein, R1 is an aliphatic hydrocarbon group having 1 to 4 carbon atoms which may have a carbon-carbon double bond, and R2 is 3 to 12 carbon atoms which may have a carbon-carbon double bond. And the total number of carbon atoms of R1 and R2 is 4 or more,
13 fatty acid or an ester or salt thereof represented by the a) less or methyl ketone general formula represented by the chemical formula _{2 R1-CH 2 -R2-CO} -CH 3 ( wherein, R1 and R2 have the same meanings as defined above), Or a monohydroxymethyl ketone represented by the general formula: R1-CH (OH) -R2-CO-CH (wherein R1 and R2 are as defined above), or a mixture thereof, A microorganism having an ability to convert to a dialcohol represented by the chemical formula 4 R1-CH (OH) -R2-OH (wherein R1 and R2 have the same meanings as described above) is converted into the above-mentioned fatty acid or a fatty acid thereof. ester or salt, wherein Mechiruketo <br/> down, or the monohydroxy ketone or cultured in a medium containing a mixture thereof, a culture of the microorganism or A treated product thereof with the fatty acid or ester or salt thereof, the methyl ketone, or the monohydroxy methyl ketone or contacting the mixture thereof in an aqueous medium, to produce and accumulate the dialcohol extracellularly, then these be collected collecting or minute is a manufacturing method of the dialcohol, wherein.

[0004] Such microorganisms may be any microorganisms as long as they have the above-mentioned conversion ability. For example, those microorganisms belonging to the genus Penicillium, Aspergillus, Fusarium, Trichoderma, Cladosporium, or a genus representing these full generations And specific strains are Penicillium decumbens IFO7
091, Aspergillus hoeni
cis) IFO 7523, Fusarium Avenasum F
SP Faber (Fsarium avenaceum f.sp. faba
e) IFO 7158, Trichoderma polysporum (Trichoderma
polysporum) IFO 9322, Cladosporium cladosporioides IFO 65
35 and the like.

[0005] The production of the dialcohol according to the present invention may be carried out by culturing the microorganism in a medium containing the precursor (hereinafter referred to as the first method). It may be carried out successively or by collecting bacteria and contacting the precursor with the precursor in an aqueous medium (hereinafter, referred to as a second method).

First, the first method will be described. These microorganisms are used as precursors for fatty acids or their esters or salts represented by the general formula 1 or methyl ketones represented by the general formula 2 A monohydroxymethyl ketone having the formula represented by Chemical Formula 3,
Alternatively, the cells are cultured under aerobic conditions in a medium containing a mixture thereof, a nitrogen source, an inorganic substance, a trace nutrient, and the like, while controlling the temperature, pH, and the like. Other carbon sources include glucose, sucrose, glycerin, dextrin,
Carbon sources commonly used for culturing filamentous fungi such as carboxymethylcellulose and kerosene may be used in combination. Nitrogen sources include ammonium sulfate, ammonium nitrate, ammonium tartrate, sodium nitrate, asparagine, peptone, corn steep liquor, etc .; inorganic substances; micronutrients: magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium chloride , Manganese sulfate, zinc sulfate, copper sulfate, ferric chloride, ammonium molybdate, potassium iodide, boric acid, various vitamins (calcium pantothenate, inositol, pyridoxine, etc.) and the like are used. The appropriate concentration of the precursor in the medium is usually 1 to 100 g / liter.

[0007] In the first method, the microorganism is cultured by a liquid culture under aerobic conditions, for example, a shaking culture method or an aeration stirring culture method. Culture is usually at a temperature of 20-35 ° C, pH
It can be carried out at 4.0 to 7.5. Culture is preferably performed until the stationary phase, and the culture time is usually 3 to 14 days.
As a result, the dialcohol represented by the chemical formula 3 can be produced and accumulated mainly outside the cells.

Next, the second method will be described. First, the microorganism used in the present invention is converted into a carbon source, a nitrogen source, an inorganic substance,
Cultivation is performed in a medium containing moderate amounts of micronutrients and the like under aerobic conditions while controlling the temperature, pH, and the like. As the carbon source, the nitrogen source, and the inorganic and micronutrients, those usually used for culturing the aforementioned filamentous fungi can be used as they are.
The culturing conditions are also the same as in the first method, but the culturing period is preferably from the latter half of the logarithmic growth phase to the stationary phase, and usually about 3 to 7 days. The culture of the microorganism thus obtained may be used as it is, or a treated product obtained by variously treating the culture, as a precursor, a fatty acid or an ester or salt thereof represented by the chemical formula 1, or a compound represented by the general formula 2 is contacted with methyl ketone represented by the formula 2, monohydroxymethyl ketone represented by the general formula 3 or a mixture thereof. As processed materials,
Examples of the concentrate include a concentrate of the culture, cells obtained by subjecting the culture to centrifugation or the like, immobilized cells, crushed cells, and an enzyme extract from the cells. The contact reaction can be carried out in any aqueous medium, but is preferably a culture solution of the bacterium to be used or a concentrated solution thereof, or a medium excluding water or a carbon source after collection (excluding the carbon source). In a suspension or the like, a fatty acid represented by the general formula 1 or an ester or salt thereof, or a methyl ketone represented by the general formula 2, or a general formula represented by the chemical formula 3 at 20 to 32 ° C. while controlling the pH to 4.0 to 7.5, more preferably 6.0 to 7.5, in the presence of the monohydroxymethyl ketone represented by No. 3 or a mixture thereof.
By reacting under shaking or aeration and stirring on a day, the dialcohol represented by Chemical Formula 4 can be mainly accumulated outside the cells. The concentration of the precursor in the reaction solution is suitably from 1 to 500 g / liter. The second method can also be carried out by continuously converting a precursor to a target substance by a bioreactor system using immobilized cells obtained by immobilizing the bacteria used in the present invention by a conventional method.

The compound used as a precursor of the target compound in the present invention is, as described above, a fatty acid or an ester or salt thereof represented by the general formula 1, a methyl ketone represented by the general formula 2 or a methyl ketone represented by the general formula 2. Is a monohydroxymethyl ketone represented by the chemical formula 3, or a mixture thereof. As described above, chemical formulas 1 to
In R3, R1 is an aliphatic hydrocarbon group having 1 to 4 carbon atoms which may have a carbon-carbon double bond, and R2 is an aliphatic hydrocarbon group having 3 to 12 carbon atoms which may have a carbon-carbon double bond. Is a monovalent aliphatic hydrocarbon group, and the total number of carbon atoms of R1 and R2 is 4 or more;
3 or less, all of which include linear or branched alkyl (alkidene), alkenyl (alkenyl), alkadienyl (alkadienyl), alkatrienyl (alkatrienyl) and the like. Fatty acids represented by the general formula 1 typically include fatty acids that form fats and oils, and include straight-chain or branched alkyl esters having 1 to 6 carbon atoms (eg, methyl ester, ethyl ester). Esters), glycerin esters such as triglyceride, diglyceride and monoglyceride can be used. Examples of the salt of the acid represented by Chemical Formula 1 include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium, and ammonium salts. R1 of methyl ketone and monohydroxymethyl ketone represented by the general formulas 2 and 3
And R2 are also as described above, and any one having such a structure can be used. In particular, using the fatty acid of Chemical Formula 1 or a derivative thereof as a raw material,
Those manufactured by the methods of Japanese Patent Application Nos. 63-320694 and 2-44386 can be suitably used.

[0010] In both the first method and the second method, the isolation and purification of the target substance from the conversion reaction completed solution can be carried out by a conventional method. That is, the target substance is extracted from the conversion reaction end solution or the cell removal solution thereof with a solvent such as chloroform, hexane, petroleum ether, and the extract is further subjected to liquid chromatography using, for example, silica gel, molecular distillation, or the like. , The desired product can be obtained.

[0011]

According to the present invention, a microorganism whose productivity has not been known is conventionally used, and a fatty acid represented by the general formula 1 or an ester or salt thereof, a methyl ketone represented by the chemical formula 2, or a chemical formula 3 Can be used as a precursor to produce an aliphatic dialcohol represented by Chemical Formula 4.

[0012]

EXAMPLES Example 1 Fsarium avenaceum fsp sp. Fabae IFO 7158
Basic medium in a 00 ml Sakaguchi flask (composition below) 100
ml, and reciprocated shaking culture (120 rp) at 28 ° C for 4 days.
m, 7 cm). Composition of medium to be used (the following is made up to 100 ml with deionized water): glucose 1.0 g dipotassium hydrogen phosphate 0.7 sodium sulfate 0.2 sodium nitrate 0.2 potassium dihydrogen phosphate 0.2 magnesium sulfate (7 (Water salt) 0.06 sodium chloride 0.05 calcium chloride (dihydrate) 0.01 vitamin preservation solution 0.1 ml mineral preservation solution 0.1 composition of vitamin preservation solution (mg / l) biotin 2 calcium pantothenate 400 Folic acid 2 inositol 2000 niacin 400 p-aminobenzoic acid 200 pyridoxine hydrochloride 400 riboflavin 200 thiamine hydrochloride 400 composition of mineral preservation solution (mg / l) manganese sulfate (4-5 hydrate) 60 zinc sulfate (7 hydrate) 300 Copper sulfate (pentahydrate) 40 Ferric chloride (hexahydrate) 250 Boric acid 60 Ammonium molybdate (4 (Water salt) 25 Potassium iodide 100 The cells are collected by centrifugation, washed with sterile water, and then transferred to 100 ml of a medium containing 1 g of methyl ketone (the above-mentioned basic medium in which glucose is replaced with methyl ketone). The cells were cultured with shaking at 4 ° C. for 4 days. 20 ml of culture termination solution
Gas chromatography (column: 5% OV-17 on Uniport HPS, 2m glass column Column temperature: 70-210 ° C, heating rate 5 ° C / mi
n Inlet and detector temperature: 230 ° C Detector: FID
Carrier gas (nitrogen) was applied, and the produced diol was measured. As a result, as shown in Table 1, an aliphatic dialcohol corresponding to each methyl ketone was obtained at a yield of about 10%.

[Table 1] の Type of methyl ketone Produced dialcohol Produced amount (mg) ─────────────────────────────────── Methylhexyl ketone 1,5-hexanediol 0.4 1,4 -Hexanediol 0.1 Methyloctyl ketone 1,7-octanediol 2.6 1,6-octanediol 7.7 1,5-octanediol 0.2 Methylnonylketone 1,8-nonanediol 4.2 1, 7-nonanediol 4.0 1,6-nonanediol 2.0 methylundecyl ketone 1,9-undecanediol 0.2 1,8-undecanediol 0.3 1,7-undecanediol 0.3 ────────────────────────────────

Example 2 Fusarium avenasum FSP Faber IFO 7158, Penicillium decumbens IFO 7091
And Cladosporium Cladosporioides IFO 6535
, Aspergillus hoensis IFO 7523 and Trichoderma polysporum IFO 9322 were inoculated in the same medium as the basal medium of Example 1 except that the amount of glucose was 0.1 g.
Reciprocal shaking culture (120 rpm, 7 cm) was performed at 28 ° C. for 3 days. Next, 100 mg of a mixture of a monohydroxy form of methyl nonyl ketone (10-hydroxy-undecane-2) was added to the culture solution.
-One 42mg, 9-hydroxy-undecane-2-one 39m
g, 8-hydroxy-undecane-2-one 19 mg) was added, and shaking culture was further continued at 28 ° C. for 1 day. Table 2 shows the results obtained by measuring the amount of dialcohol produced in the culture solution in the same manner as in Example 1.

[Table 2] 菌 Total nonanediol production of the strain ──── ─────────────────────────────── Fusarium Avenashum F 2.3 mg SP Faber IFO 7158 Penicillium Decumbens IFO 7091 0.5 Cladosporium 0.7 Cladosporioides IFO 6535 Aspergillus hoenisis IFO 7523 0.2 Trichoderma polysporum IFO 9322 0.2 ───────────────── ──────────────────

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12P 7/18 C12R 1: 885) (C12P 7/18 C12R 1: 885) (C12P 7/18 C12R 1: 645)

Claims (1)

(57) [Claims] 1. A compound represented by the general formula:₂-R2- (CH₂)₂-COO
H (wherein, R 1 is a carbon which may have a carbon-carbon double bond.
An aliphatic hydrocarbon group of Formulas 1 to 4, R2 is a carbon-carbon double bond;
Divalent aliphatic hydrocarbons having 3 to 12 carbon atoms which may have union
A total number of carbon atoms of R1 and R2 is 4 or more,
13 or less) or a fatty acid ester thereof
Or a salt or a compound represented by the general formula:₂-R2-CO-CH₃  (Wherein, R1 and R2 are as defined above)
Methyl ketone, or a compound represented by the general formula: R1-CH (OH) -R2-CO-CH₃  (Wherein, R1 and R2 are as defined above)
Monohydroxymethyl ketone or a mixture thereof
The general formula is represented by Chemical Formula 4 R1-CH (OH) -R2-OH (wherein, R1 and R2 are as defined above).
Has the ability to convert to dialcohol, Penicillium,
Aspergillus, Fusarium, Trichoderma or
Is a microorganism belonging to the genus Cladosporium,
Or its esters or salts, the aforementioned methyl ketones, none
The monohydroxymethyl ketone, or their
Culture in a medium containing the mixture or culture of the microorganism
Or the treated product and the fatty acid or ester thereof
Or the salt, the methyl ketone, or the monohydroxy
Cimethyl ketone or a mixture thereof in an aqueous medium
To produce and accumulate the dialcohol outside the cells.
And then collect or sort them.
The method for producing the dialcohol described above.