JPH04148688A - Production of methyl ketone - Google Patents

Abstract

PURPOSE:To obtain a methyl ketone in high yield, free from chemical odor, useful as a flavor, etc., for dairy products, soaps, etc., by contact of a fatty acid, etc., with the cell membrane wall of specific microorganisms in the presence of divalent alkaline earth metal ion. CONSTITUTION:The objective compound of formula II can be obtained by contact of (A) a fatty acid of formula I (A is aliphatic hydrocarbon which may have carbon-carbon double bond) or its ester or salt with (B) the cell membrane wall of microorganisms capable of converting the compound A to the objective methyl ketone of the formula II (e.g. Penicillium decumbens IFO 7091) in the presence of (C) 0.1-100mM of divalent alkaline earth metal ion (e.g. Mg<2+>, Ca<2+>).

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing a corresponding methyl ketone from a fatty acid or an ester or salt thereof, or a mixture thereof, using an enzyme present in the cell membrane wall of a microorganism. Methyl ketone can be used as a flavor for dairy products, soaps, etc., or as a solvent for fragrances, dyes, etc.

(Prior art and problems to be solved by the invention) Conventionally,
Methyl ketones are mainly produced by chemical synthesis, but such products retain a so-called medicinal odor, which is particularly problematic in the fragrance industry. For this reason, production methods using microorganisms are attracting attention. These methods include a method using bacteria as a raw material for re-alkanes, a method using yeast to produce re-alkane-2-ol, and a method using filamentous fungi to produce fatty acids. It has also been proposed in Japanese Patent Application No. 63-320694.

Although methods using such microorganisms do not have the above-mentioned drawbacks of chemical synthesis methods, they do have problems such as poor yields because part of the substrate is consumed for the growth of microorganisms, and difficulty in continuous production. There was a point.

Therefore, attempts have been made to immobilize yeast-derived enzymes and yeast and contact them with corresponding secondary alcohols, but this method is not practical because the raw material alcohol is expensive.

(Means for Solving the Problems) The present inventors have constructed a cell membrane wall of a microorganism that belongs to the genus Penicillium and has the ability to convert fatty acids or their esters into the corresponding methyl ketones, using divalent alkaline earth metal ions. We have discovered that methyl ketones can be obtained in high yield by contacting the fatty acids etc. in the presence of
While further researching, it was discovered that there are many microorganisms other than the genus Penicillium that have the ability to produce methyl ketones in this manner, and the present invention was completed.

That is, the present invention provides a fatty acid represented by the general formula % (wherein A represents an aliphatic hydrocarbon group which may have a carbon-carbon double bond) or an ester or salt thereof, The cell membrane wall of a microorganism obtained from a microorganism having the ability to convert into methyl ketone represented by the general formula % formula % (wherein A has the same meaning as above) was prepared in the presence of divalent alkaline earth metal ions. in,
A method for producing methyl ketone, which comprises contacting the fatty acid, an ester or salt thereof, or a mixture thereof.

The microorganisms include, for example, Penicillium, Trichoderma,
Aspergillus, Fusarium, Hypophlea, Cladosporium, Neosartruya, Hemicarpenteres, Caetosartruya, Gibberella, Mycosphaerella, Eupenicillium, Emericella, Monascus, Syncephalastrum, Bodostroma genus,
Hamigera, Trichoderma, Pheneria, Pleusia, Microascus, Tamaromyces, Sucrea
Any microorganism may be used as long as it belongs to the genus Cleista, Benicilliopsis, Dichotomyces, or Aureobasidium and has the above-mentioned conversion ability.

As a member strain, Penicillium decumbens (Pe
nicilliua decumbens) IPo
7091. ) Lycoderma hamatum (Tricho)
deraa 71FO31291゜Trichoderma polisvorum (T, emetic) IFO9322, Aspergillus v.
entti) rFo 8864, Aspergillus terreus (A, terreus) HUT 2099,
Fusarium scilla, nee (Fsarius 5olan)
i) HUT 5013, Fusarium semitictum (
F, semitectus) IPO30926, Fusarium avenaceus (F, avenaceus)
) IFO7158, Fusarium oxysporum (F
, oxysporus) HUT 5012, Hypocreanigricans CHypocreaanigricans
s) IFO30611, Cladosporium cladosporioides (CIadosporius+ clad
osporioides) IFO6535, Neosartorya ficieri (Neosartorya ris)
heri) HUT 4106, hemical penterus
Acanthos boras (llemicarpenteles)
acanthosporus) IFO9490, Chaetosporya stromatoides CChaetosar
torya sLromatoides) IFO965
2. Gibberella lateritium
laeteritium) IFO7705, Mycosphaerella 0 melonis (Mycosphaerell
a melonis) IFO8776, Eupenicillium javanicum (Eupenieilliuw + j
avanicus+) IFO7999, Nectria・
Nectria flammea IFO
9628, Emericella nidulans (Eserice)
lla n1dulans) IFo 863G, Monascus anka (Monascus anka) HUT
4011, Syncephalastrum racemosum (Syn
cephalastrus racemosus) F
IUT 1300, Bodostroma codicesbus (
Podostroma cordyceps) IFO
9019, Hasigera striata (Hasigera 5
triata) IFO6106, Trichochoma paradoxa 5, Microascus desmosporus (Microascus desmosporus)
cusdesI+1osporus) IFo 67G
+, Tamaromyces φ emersonii (Tararom
yces emersonii) IFO31126,
Scleroclei ornata (Scleroclei)
staornata) IFO4042, Penicilliopsis clavalierformis (Penicilliops)
IFO67
64, Dichotomyces segebi
myces cejpii) HUT 4011.

In the present invention, microorganisms having the above-mentioned conversion ability are cultured and propagated by a known method, and if necessary, the bacteria are collected and washed by an appropriate means such as centrifugation, and then subjected to ultrasonic waves, glass peas, French press, etc. The bacterial cells are disrupted by a known method. The crushed material is dissolved in water by 11%, 500-5000 x
G, more preferably 1 to 2000 xG
Centrifuge for 30 minutes and collect the precipitated cell membrane wall of the microorganism. If necessary, the precipitate can be resuspended in water for washing and centrifugation can be repeated under the same conditions.

In order to convert fatty acids etc. into methyl ketones using the cell membrane wall, the substance should be adjusted to pH 5 to 8, more preferably pH 6.5.
~7,5 in an aqueous solvent, based on the amount of protein.
Suspend in a range of 1-10 g/z (l), add fatty acid and/or MW fatty acid salt, or fatty acid ester as a substrate, and stir at 15-50°C, preferably 25-1
React at 35°C. Although a wide range of fatty acids having 4 to 20 carbon atoms can be used in the present invention, so-called medium-chain fatty acids having about 6 to 12 carbon atoms are more suitable in terms of reactivity.

In the present invention, the presence of alkaline earth metal ions such as calcium and magnesium in the reaction system is an essential requirement, and the concentration thereof is approximately 0.1=100 akl.

The reaction is carried out at a temperature of 0.5 to 24
Do time.

In addition, in the present invention, in addition to carrying out the reaction in a solution as described above, the cell membrane wall is packed into a column, and while the column is maintained at an appropriate temperature, a substrate dissolved or suspended in water or a buffer solution is added. It can be made to pass continuously.

The target substance, methyl ketones, can be isolated and purified from the reaction system by the conventional method described in, for example, Japanese Patent Application No. 63-320694.

(Effects of the Invention) According to the present invention, methyl ketones can be enzymatically obtained in high yield using inexpensive fatty acids as raw materials. By immobilizing the cell membrane wall, it is also possible to continuously produce methyl ketone.

(Example) Examples are shown below.

Example 1 Penicillium decumbens IFo 7091 and Fusarium solani HUT 5013 were inoculated into 1% glucose basal medium 100xff and cultured with shaking at 28°C for 4 days. The obtained bacterial cells were collected by suction filtration through No. 2 filter paper, dispersed in 2001M Tris-HCl buffer (pH 7.5), and crushed using glass beads (0.4xz).

The liquid obtained by suction filtration with No. 2 filter paper is 1,500x
The mixture was centrifuged at G for 10 minutes, and the precipitate (cell membrane wall) was collected.

Cell membrane walls were washed twice with the same buffer and centrifugation was repeated.

A cell membrane wall with a protein content of 0.6x9 was taken,
113 μg of sodium caprate (100 μ9 as capric acid) was dispersed in 20 oz IM Tris-HCl buffer (pH 7,5) 1 zQ and held at 28° C. for 4 hours.

In addition, tests were conducted in which calcium chloride or magnesium chloride was added to the reaction system in an amount of IOzM.

After a period of time, 1 to 2 drops of 6N hydrochloric acid were added to stop the reaction, and the chloroform extract was quantified using gas chromatography for methyl ketones and HPLC (high performance liquid chromatography) for fatty acids as PNBDI derivatives. The results are shown in Table-1.

As is clear from Table 1, the reaction system? In the absence of alkaline earth metal ions, the amount of methyl ketone produced was only about 1 to 2% of the substrate, but by adding the same ions, the amount produced was 5 to 10 times greater.

Example 2 Using the cell membrane wall of Penicillium decumbens obtained in Example 1, a reaction was carried out using the fatty acids shown in Table 2 (100 μm of fatty acid was used in each case) as a substrate. The procedure was the same as in Example I except that the calcium ion concentration in the reaction system was IOIM and the reaction time was 16 hours.

(Space below) The results are shown in Table 2. The amount of methyl ketone produced from fatty acid salts is highest from capric acid (10 carbon atoms), followed by caprylic acid (8 carbon atoms) and caproic acid (6 carbon atoms).
), lauric acid (12), and myristic acid (12).
The amount produced from 14) was small.

Example 3 Using cell membrane walls obtained by treating the microorganisms shown in Table 3 in the same manner as in Example I, the calcium ion concentration in the reaction system was adjusted to 10zM.
The mixture was reacted at 28° C. for 4 hours.

The substrate for each is sodium caprate 113μ
f (100 μg as capric acid) was used.

The details of the operation were the same as in Example 1.

The results are shown in Table 3.

(Leaving space below) Table 3 Trichoderma, Trichoderma hamatum, Aspergillus polyborum, Aspergillus wentii, Fusarium terreus, Fusarium semitictum, Fusarium avenaceum, Fusarium oxysporum, Hibocrea nigricans, Cladosporium nigricans, Cladosporio. Ides neosartorya ficieri Hemicarpenteres acanthosvorascaetosartruya stromatoides IFO 9652 Gibberella lateritium IPo 7705 Mycosphaerella melonis IFO 8776 Euvenicillium japanicum IFo 7999Ht
lT 4106 IFO31291 1FO9322 IFO8864 HUT 2099 IFO30926 IFO7158 HUT 5012 IFO30611 1FO6535 IFO9490 9,45 1? , 54 11.17 8.53 26.75 7.00 0.17 24.75 18.60 18.79 12.5g 9.67 8.80 2.97 Table-3 (2) Nectria flamea IFo 9628
Emericella nidulans IFo 863
0 Monascus anchor HUT 401
1 Synthesphalastrum racemosum HUT 130
0 Bodstrom Codycebus IFo 901
9 Hamigera striata IFo 61
06 Trichochoma paradoxa IFo 67
65 Fueneria flavibes IFo 40
52 Pleusia isomella HUT 41
45 Microascus desmosporus IFo 67
61 Tamaromyces emersonii IFo 31
126 Sclerocleista ornata IFo 4
042 Penicilliopsis Clavalierformis IFO6
764 Deicotomyces cegepi-11UT 401
1 Aureobasidium sp. FERM P-
112772,68 1,18 0,91 O04I O035 0,06 0,05 0,06 0,04 0,05 0,04 0,04 0,03 0,03 1,41 Patent applicant Showa Sangyo Co., Ltd.

Claims (1)

[Claims] Fatty acid represented by the general formula ▲ Numerical formula, chemical formula, table, etc. ▼ (wherein A represents an aliphatic hydrocarbon group which may have a carbon-carbon double bond) or its ester or salt, into a methyl ketone represented by the general formula ▲ Numerical formula, chemical formula, table, etc. ▼ (wherein A has the same meaning as above). wall in the presence of divalent alkaline earth metal ions,
A method for producing methyl ketone, which comprises contacting the fatty acid, an ester or salt thereof, or a mixture thereof.