JPH03247290A - Production of hydroxyalkanone by fermentation method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing hydroxyalkanones from alkanones or alkanoic acids or derivatives thereof by the action of microorganisms. Hydroxyalkanones can be used as fragrances and synthetic raw materials.

(Problems to be solved by conventional techniques and inventions) Conventionally,
As a method for producing methyl ketone from fats and oils using microorganisms, Penicillium roqueforti (Penicillium
A method using microorganisms belonging to the iuw dish ■1 Rainbow Dan) (
R, C, Lawrence et al., J, Gen.

Microbiol, 51.289-302 (1
968), and R, D. King et al., J. Sci.
Food Agric, 30.197-202 (
1979)), Penicillium palitans (P.
ans) Moji < is Penicillium glocum (P,
A method using microorganisms belonging to Shinanami L) [W, N, 5t
okoe, Biochem, J.

22, 8O-93 (1928)), Penicillium
Method using microorganisms belonging to caseicolum (P, caseicolu+wn) (T, lamberet et al., Rev. La1tiere Fr, 406 +13
4 (1980) ), Penicillium citrinum (P
, citrinu+Il) C old 298303 and C
I'lI 298309, or Eur
method using microorganisms belonging to the genus otiu+w) (J,
L., Kinderler et al.

Phytoche+5istry 23.2847-
Phyt.
ochemistry 26.1417-1420 (
1987)), Aspergillus niger (A, Dan L.
L) or a method using microorganisms belonging to Aspergillus fumigatus (A, 1 and 5 tarkl) (M, 5 tarkl)
e, Biochem, Z.

anus, 371-463 (1924)), Trichoderma viride
A method using microorganisms belonging to (J, Ferment, T.
echnol, 66403-407.1988), a method using microorganisms belonging to the genus Trichoderma (Yagi et al., Abstracts of the 1988 Japan Fermentation Engineering Conference (10/10 issue) 6 and 1179-11), Penicillium genus,
A method using microorganisms belonging to the genus Aspergillus, Fusarium, or Trichoderma (Journal of the Japanese Society of Agricultural Chemistry,
3), 350.1989) is known.

Furthermore, a method for producing an oily substance having an apple-like aroma from fats and oils using microorganisms belonging to the genus Rhizopus or the genus Mucor is known (Japanese Unexamined Patent Publication No. 57-208991).

In addition, in a miscellaneous article on the production of hydroquinone from phenol by Mycobacterium bacteria, methyl ketone is assimilated as a carbon source to increase hydroquinone production activity.
-Hydroxy-2-butanone and 4-hydroxy-2-
It has been reported that it produces butanone (BIOI
NDLISTRY 7 (2).

106-109 (1990)).

However, the extracellular production of corresponding hydroxyalkanones from longer chain alkanones by fungi is unknown.

Furthermore, there is no known method for producing hydroxyalkanones from alkanoic acids or derivatives thereof using microorganisms.

[Means to solve the problem]

The present inventors have discovered that among various microorganisms there are microorganisms that produce hydroxyalkanones using alkanones, alkanoic acids, or derivatives thereof as precursors, and have completed the present invention. That is, the present invention belongs to the genus Fusarium, Penicillium, Aspergillus, Trichoderma, or Thallatosborium, and has the general formula (1) % formula % (1) (wherein R1 represents an alkyl group having 5 or more carbon atoms, R represents a methyl, ethyl or propyl group). 3.4 or 5th carbon represents the corresponding R1 group hydroxylated, R has the same meaning as above) and/or the general formula (I
I) Rg-CH2-CH! -C-OH(If)1 (in the formula, R2 represents an alkyl group having 5 or more carbon atoms) or its ester or salt represented by the general formula (IV) R4CCH3(IV)1 (in the formula, R4 represents the corresponding R2 group in which the 2.3.4 or 5th carbon is hydroxylated from the ω position to the ω position)
A microorganism having the ability to convert into a species is cultured in a medium containing the alkanone corresponding to the conversion ability, or the alkanoic acid or an ester or salt thereof, or a mixture thereof, or a culture of the microorganism or a treated product thereof and the contacting the alkanone, or the alkanoic acid or ester or salt thereof, or a mixture thereof in an aqueous medium,
A general formula (which is characterized by producing and accumulating at least one hydroxyalkanone of the general formula (I) or at least one hydroxyalkanone of the general formula (IV) outside the bacterial cells, and then collecting it) A method for producing at least one hydroxyalkanone of DI) or at least one hydroxyalkanone of general formula (IV) is provided.

The microorganism used in the present invention belongs to the genus Fusarium, Penicillium, Aspergillus, Trichoderma, or Cladosporium, and may be any microorganism having the above-mentioned conversion ability.

A specific bacterial strain is Fusarium avenaceum (Fu
sarium avenaceum) IFO71
58, Fusarium scilla'- (F, 5olani)
HUT 5013 (IFO5232), Penicillium decu
mbens) IFO7091, Penicillium paritans (P, for Danden) IFO8801, Aspergillus wenti (Aser 1llus we
ntii) IFO8864, Aspergillus IFO 7523, Trichoderma borisporum (T
richoderma 赳n且HK憇) IFO 93
22, Cladosporium cladosporioides (CIad■■d”-clados orioide
S) IFO 6535 is mentioned.

The production of hydroxyalkanone in the present invention may be carried out by culturing the above-mentioned microorganisms in a medium containing a precursor (hereinafter referred to as the first method), or by culturing the microorganisms in a conventional medium and subsequently or by harvesting the microorganisms. It may also be carried out by contacting with a precursor in an aqueous medium (hereinafter referred to as the second method).

First, regarding the first method, these microorganisms are treated with an alkanone of general formula (1) as a carbon source and precursor, an acid of general formula (II) or an ester or salt thereof, or a mixture thereof, a nitrogen source, an inorganic substance, In a medium containing moderate amounts of micronutrients, etc., under aerobic conditions, the temperature
Cultivate while adjusting pH etc.

Other carbon sources that are commonly used for culturing filamentous fungi, such as glucose, sucrose, glycerin, dextrin, carboxymethyl cellulose, and kerosene, may also be used in combination. Nitrogen sources include ammonium sulfate, ammonium nitrate, ammonium tartrate, sodium nitrate, asparagine, peptone, corn staple liquor, etc. Inorganic substances and micronutrients include 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.) are used. Furthermore, in order to avoid contamination by bacteria during culture, a bactericidal substance such as chloramphenicol may be contained in the medium.

The concentration of the above precursor in the medium is 1 to 500 g/i!
.

is appropriate. In addition, the precursor concentration is 50 to 500 g/l
When carried out, there is an advantage that the target product can be recovered by a simple purification method as described below.

The target product-producing strain in the first method may be cultured under aerobic conditions by a liquid culture method, such as a shaking culture method or an aerated agitation culture method. Culture is usually carried out at a temperature of 20-32°C and a pH of 5.
, 0 to 9.0. The culture is preferably carried out until the stationary phase, and the culture time is usually 3 to 14 days. This allows at least one hydroxyalkanone of the corresponding general formula (I[[) or at least one hydroxyalkanone of the corresponding general formula (IV) to be accumulated mainly outside the bacterial cells.

Next, regarding the second method, first, the microorganism used in the present invention is cultured in a medium containing appropriate amounts of carbon source, nitrogen source, micronutrients, etc. under aerobic conditions while controlling temperature, pH, etc.

Carbon sources include glucose, sucrose, glycerin,
Dextrin, carboxymethyl cellulose, soybean oil,
In addition to carbon sources commonly used for culturing filamentous fungi such as palm kernel oil and kerosene, alkanones of general formula (1), acids of general formula (n), esters or salts thereof, or mixtures thereof are used. As the nitrogen source, inorganic substances and micronutrients, the same ones as in the first method are used. Bactericidal substances similar to those in the first method may also be used.

The culture in the second method is usually carried out at a temperature of 20-32°C, p) I
5.0 to 9.0 using a liquid culture method. Culture is preferably carried out from the latter half of the logarithmic growth phase to the stationary phase, and the culture time is usually 3 to 7 days.

The alkanone of the general formula (1), or the fatty acid of the general formula (It) or its ester or salt or contact with a mixture thereof. Processed products include culture concentrate,
Examples include bacterial cells obtained by subjecting a culture to centrifugation, immobilized bacterial cells, and enzyme preparations extracted from bacterial cells.

The contact reaction can be carried out in any aqueous medium, but it is preferable to use a culture solution of the bacteria to be used, its concentrate, bacterial collection condensate, or a medium from which a carbon source has been removed (other than the one used in the previous step except for the lack of a carbon source). The alkanone of general formula (I), the fatty acid of general formula (II) or its ester or salt, or a mixture thereof is added to a suspension in a medium similar to that used for culture, and the pH is adjusted. 5.0-9.
At least one of the corresponding hydroxyalkanones of the general formula (I[I) is reacted at 20 to 32°C for 1 to 7 days with shaking or aeration while controlling the temperature to 0.
Alternatively, at least one hydroxyalkamono of the corresponding general formula (IV) can be mainly accumulated outside the bacterial cells. The concentration of the above precursor in the reaction solution is 1 to 500
g/j2 is appropriate. In addition, considering the refining aspect, the first
For the same reason as in the method, 50 to 500 g/l is preferable.

The second method can also be carried out by continuously converting the precursor into the target product using a single bioreactor system using immobilized bacterial cells obtained by immobilizing the bacteria used in the present invention by a conventional method.

As mentioned above, the compound used as a precursor of the target compound in the present invention is an alkanone represented by the general formula (1), an alkanoic acid represented by the general formula (II), an ester or salt thereof, or a mixture thereof. .

In general formula (1), R1 is an alkyl group having 5 or more carbon atoms, but due to the yield of the target product, the number of carbon atoms is 6 to 18.
It is preferable that it is, it is more preferable that it is 6-13, it is still more preferable that it is 6-11, and it is 7-9.
Most preferably. Although such an alkyl group may be branched, it is preferably straight.

Specific examples of R1 include n-pentyl group, n-hexyl group, n-heptyl group, n-octyl group, n-nonyl group, n-
-decyl group, n-undecyl group, n-dodecyl Ln-tridecyl L n-pentadecyl group, n-octadecyl group and the like.

Specific examples of the alkanone of general formula (1) include 2-heptanone, 2-octanone, 3-octanone, 2-nonanone, 3-nonanone, 4-nonanone, 2-decanone, 3-decanone, 4-decanone, and 2-undecanone. , 3-undecanone, 4-undecanone, 2-dodecanone, 3-dodecanone, 4-dodecanone, 2-tridecanone, 3-tridecanone, 4-tridecanone, 3-tetradecanone, 2-
Examples include pentadecanone, 4-pentadecanone, 2-eicosanone, and the like.

In general formula (n), R2 is an alkyl group having 5 or more carbon atoms, preferably 6 to 15 carbon atoms, and 6 to 15 carbon atoms.
-11 is more preferable, and 6 or 7 is most preferable. Such alkyl groups may be branched, but are preferably linear.

Specific examples of R2 include n-pentyl group, n-hexyl group, n-hebutyl group, n-octyl group, n/=/L4, n
-decyl group, n-undecyl group, n-dodecyl group, n-
) lysosyl group, n-pentadecyl group, etc.

-IIIQ As the alkanoic acid ester of formula (II), this microorganism hydrolyzes it in its metabolic system to form the general formula (rV)
Although there is no particular limitation as long as the ester can produce a hydroxyalkanone of
Diglycerides and monoglycerides may also be used. Specific examples of dark glycerin esters include tricaprylin (triglyceride of caprylic acid), tricaprin (triglyceride of capric acid), trilaurin (triglyceride of lauric acid), trimyristin (triglyceride of myristic acid), and tribalmitin (triglyceride of valmitic acid). ), tristearin (triglyceride of stearic acid), mono- or cicaprine (mono- or diglyceride of capric acid), mono- or dilaurine (mono- or diglyceride of lauric acid), and the like. The ester of the alkanoic acid of general formula (n) may also be an alkyl ester, especially a linear or branched alkyl ester having 1 to 6 carbon atoms (for example, methyl ester, ethyl ester, etc.), hensyl ester, etc. good.

Specific examples of such esters include methylcaprin (methyl caprate), ethylcaprin (ethyl caprate).
, methyllaurin (methyl laurate), ethyllaurin (ethyl laurate), and the like.

-I'1 Salts of the alkanoic acid of formula (II) include salts with alkali metals, such as sodium salts, potassium salts, salts with alkaline earth metals, such as calcium salts, ammonium salts, and salts with amines. It will be done.

The precursor used in the present invention is an alkanoic acid of general formula (n),
It may also be a mixture of two or more of these esters or salts. That is, it may be a combination of acids, esters, salts, acids and esters, esters and salts, or a combination of acids, esters, and salts. The most common mixture used in the present invention is oil, which may be either vegetable oil or animal fat. Vegetable oils include coconut oil, palm oil, palm kernel oil, evening hair oil, camellia oil, olive oil, castor oil, sesame oil, rapeseed oil, cottonseed oil, soybean oil, corn oil, peanut oil, safflower oil, oleic oil, Grapeseed oil and the like are used, and as the animal fat and oil, milk fat (butter fat), lard, tallow, beef kenneen oil, etc. are used.

In both the first method, the second method, and the compounds of general formulas (1) and (II), the hydroxyl group is substituted at the 2, 3, 4 or 5 carbon position from the ω position to the ω position. Although this occurs for only one of them, it does not always occur at a fixed level for each molecule, and usually two or three types of target substances are produced at the same time. That is, when the number of carbon atoms in RI or R2 is relatively short, two types of target products in which the carbons at the 2nd and 3rd positions are each monohydroxy-substituted are produced simultaneously, and R
, or 2.3.4 when the number of carbon atoms in R2 is relatively long
and two or three types of target products in which the carbon at the 5th position is substituted with monohydroxy are co-produced.

For example, when Fusarium ahenacium IFO7158 is used, if R1 is 8 or less, two types of target products are mainly produced, in which the carbons at the 2nd and 3rd positions are monohydroxylated, and in some cases, the target product is monohydroxylated at the 4th position. It also produces small amounts of substances. When R2 is from 9 to about 1O, three types of target products are produced, each monohydroxylated at the 2.3 and 4 positions, and when R1 is about 10 or more, three types are produced, each monohydroxylated at the 3.4 and 5 positions. The target object is generated.

In both the first method and the second method, the target substance can be isolated and purified from the conversion reaction completed liquid by a conventional method. That is, the target product (usually a mixture) is extracted from the conversion reaction completed liquid or its bacterial cell removal liquid with a solvent such as chloroform, hexane, petroleum ether, etc., and the extract is subjected to column separation operation such as liquid chromatography or silica gel column chromatography. The target product can be obtained in solid form by subjecting it to water and then distilling it. Furthermore, steam distillation may be incorporated into the initial stage of purification by taking advantage of the volatility of the target compound.

Note that even when the amount of precursor is increased and oil droplets are present in a layered manner on the surface of the culture solution, the desired product can be sufficiently produced. If such conditions are adopted, instead of solvent extraction after culturing,
Advantageously, the product can be recovered by easier means such as centrifugation. As mentioned above, an appropriate amount of such an advantageous precursor is 50 to 500 g/l.

〔Example〕

Next, examples of the present invention will be shown.

Microorganisms shown in Table 1 above were placed in 5 test tubes with a diameter of 18IllI11.
Inoculate the following tricaprin medium (pH 7, 2) in d,
The cells were cultured with shaking at 8°C for 5 days.

Used medium composition: Nitricaprin 1.01 dNa
NO+ 0.2g (
NH4) 2504 0.
2gK2HPO40,7g KH,PO40,2g Gate gso4・7H200,06g CaCIz' 2Hz0 0
．． 01gNaCl
0.05g-vitamin preservation solution 0
．． 1 bottle mineral preservation solution 0.1d1
% Chloramphenicol 0.21 d in deionized water 100 Composition of vitamin preservation solution (mg/1) - Biotin 2 Calcium pantothenate 400 Folic acid
2 inositol 2
000 Niacin 400p-
Aminobenzoic acid 200 Pyridoxine hydrochloride 400 Riboflavin
200 Thiamine Hydrochloride
Composition of 400 mineral preservation solution (mg/l) ns
O+' 4 ~5H2060ZnSO4・7H2
0300 CuSOa ・5Hz0 40F
eC1:+ ・68zO250 1(3BO360 (NH4)6 MO7024・4H2O25Kl
100 ml of cultured solution
The liquid extracted with R1 with chloroform was subjected to gas chromatography (column: 5% 0V-17 on Uniboat tIPS).
, 2m glass column Column temperature = 70-170°C15
°(: /sin temperature increase Inlet detector temperature: 230℃
Detector: FID Carrier gas: Target products 2-nonanone and monohydroxy-2-
The production amount of nonanone was measured. The results are shown in Table 1.

Table 1 The above monohydroxy-2-nonanone is the result of the above gas chromatography in the case of any microorganism: 8-hydroxy-2-nonanone: 7-hydroxy-2-nonanone:
It was composed of 6-hydroxy-2-nonanone-about 1:1:0.1 (weight ratio).

Fruit 1111 Same medium as Example 1 (p87.2) 100i except that it contains 0.1 g of glucose instead of Tricaprin 1.0-
d was placed in a 500-meter round flask, inoculated with 5 x 10' Fusarium avenacium IF07158 spores, and cultured with shaking at 28°C for 3 days. Various ketones low+g shown in Table 2 were added to the cultured solution, and the mixture was further cultured at the same temperature for 1 to 3 days.

The final culture solution was treated and analyzed in the same manner as in Example 1. The results are shown in Table 2.

Table 2 All hydroxyalkanones produced are counted from the ω position farthest from the oxo position of the raw material alkanone, including the ω position.
3. Fewer carbons at the 4th and 5th positions (both were hydroxylated; i.e., in the case of using 2-tridecanone, three types of monohydroxy carbons were hydroxylated at the 3rd, 4th, and 5th positions). Alkanes were produced, and the produced weight ratio was approximately 1:1:l.In addition, in the case of using 2-undecanone, three types of monohydroxyalkanes in which the carbons at the 2-, 3-, and 4-positions were hydroxylated were produced. canon is generated,
The product weight ratio was approximately 1:1:0.5. Also 2-
When decanone and 2-nonanone are used, three types of monohydroxy alkanones in which the carbons at the 2-, 3-, and 4-positions are hydroxylated are produced, and the weight ratio of the products is approximately 1: When using 3:0.1.2-nonanone, the ratio was approximately 1:1:0.1. Furthermore, in the case of using 2-octanone and 3- or 4-decanone, the carbons at the 4th and 5th positions are not hydroxylated, and the weight ratio of monohydroxyalkanes produced with hydroxylated carbons at the 2nd and 3rd positions is approximately 2=1.

Working group The operation was carried out in the same manner as in Example 2 except that 2-nonanone was used as the main alkanone and the microorganisms shown in Table 3 were used as the microorganisms.

The results are shown in Table 3.

Table 3 The above hydroxy-2-nonanone is 8-hydroxy-2-nonanone: 7 hydroxy 2
-nonanone: 6-hydroxy 2-nonanone - about 1:1:
0.1 (weight ratio).

The procedure was carried out in the same manner as in Example 2 except that 2-undecanone was used as the alkanone and the microorganisms shown in Table 4 were used as the microorganisms.

The results are shown in Table 4.

Table 4 The above hydroxy-2-undecanone is 10-hydroxy-2-undecanone:9-hydroxy-2-undecanone28-hydroxy-2-undecanone-approximately 1:1:0.5 (by weight) in the case of any microorganism. ratio).

Except for using palm kernel oil (produced in Malaysia > 1.0 d) instead of tricaprin 1.0 d as the carbon source/precursor.
Cultivation was carried out in the same manner as in Example 1, and the hydroxyalkanone production results shown below were obtained.

Hydroxyalkanone production results (-g) Hydroxy-2
-nonanone 0.01 hydroxy-2-undecanone 0.50 hydroxy-2-tridecanone
0.01 In the above, hydroxy-2-tridecanone is ω
It is a mixture of carbons in which the 3.4th or 5th carbon position is hydroxylated, and the ratio is approximately 1:1:1. are hydroxylated in a ratio of approximately 1:1:0.5;
2-nonanone is a mixture of carbons hydroxylated from the ω position to the 2.3 or 4 position, and the ratio is approximately 1:1.
:0.1.

Fusarium.
Ahenacium IFO7158 was inoculated and cultured with shaking at 28°C for 4 days, followed by centrifugation and washing with sterile water.

10g of 2-undecanone instead of 1.0d of tricaprin
The same medium 32 as in Example 1 containing 51! , and inoculated with all the bacterial cells obtained above. 28°C
2 days at a culture temperature of 0.51/-in, agitation 2
Cultured at 5 Orpm.

After culturing, the cells were suction-filtered using No. 2 filter paper to remove bacterial cells.

The bacterial cells were washed with hot water at 80°C, and the washing liquid was combined with the filtrate. The filtrate was centrifuged to obtain 93g of oil layer, which was then added to 5n+
The mixture was heated to 125° C. under a vacuum of +nHg and separated into a 64 g fraction and a 27 g residue. The residue is monohydroxy-2-undecanone with a purity of 80/purity, and those substituted at the 2-, 3-, and 4-positions from the ω position are X:1:O.

5 (weight ratio).

〔Effect of the invention〕

Fermentation production of a hydroxyalkanone of general formula (III) using an alkanone of general formula (1) as a precursor, which has not been known until now, and an alkanoic acid of general formula (II), an ester or salt thereof, or a mixture thereof Fermentative production of hydroxydialkylmethylketone of general formula (IV) using as a precursor is carried out by specific filamentous fungi.

Claims (1)

[Scope of Claims] 1. Belongs to the genus Fusarium, Penicillium, Aspergillus, Trichoderma or Cladosporium, and has the general formula (I) ▲Mathematical formula, chemical formula, table, etc.▼(I) (In the formula, R_1 represents an alkyl group having 5 or more carbon atoms, and R
represents a methyl, ethyl or propyl group) is represented by the general formula (III) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(III) (In the formula, R_3 is 2, including the ω-position, Corresponding R_1 hydroxylated at 3rd, 4th or 5th carbon
and/or the general formula (II) ▲There are numerical formulas, chemical formulas, tables, etc.▼(II) (in the formula, (R_2 represents an alkyl group having 5 or more carbon atoms) is an alkanoic acid or its ester or salt represented by the general formula (IV) ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ (IV) (In the formula, R_4 is the ω position corresponding R_2 in which the 2nd, 3rd, 4th or 5th carbon including the ω position is hydroxylated
A microorganism having the ability to convert at least one type of hydroxyalkanone represented by (representing a group) is cultured in a medium containing the alkanone corresponding to the conversion ability, the alkanoic acid, its ester or salt, or a mixture thereof. Alternatively, by contacting a culture of the microorganism or a treated product thereof with the alkanone, the alkanoic acid, its ester or salt, or a mixture thereof in an aqueous medium, the hydroxyl of general formula (III) is released from the microorganism. At least one kind of alkanone or at least one kind of hydroxyalkanone of general formula (IV) is produced and accumulated, and then collected, at least one kind of hydroxyalkanone of general formula (III) or general A method for producing at least one hydroxyalkanone of formula (IV).