JPH0634732B2 - Method for producing unsaturated wax ester - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a method for producing an unsaturated wax ester from a fat and oil in a high yield in a microbiological manner.

Wax esters are chemically esters of higher aliphatic alcohols and higher fatty acids, and have the effects of protecting the epidermis of animals and plants, maintaining moistness, and preventing heat loss. It is used in large quantities as a raw material for cosmetics, water repellents and cosmetics.

Conventionally, most unsaturated wax esters are produced from natural products derived from animals and plants, and the supply thereof has not always been stable. In particular, sperm whale oil has produced wax esters suitable as raw materials for high-quality lubricating oils and cosmetics, but in recent years, there has been a need for an alternative raw material due to supply anxiety caused by the prohibition of whaling.

Sperm whale oil is a straight-chain ester composed of a long-chain fatty acid having a carbon number of 14 to 18 and a long-chain alcohol, and the fatty acid and alcohol are saturated or
It is unsaturated having one double bond.

As an alternative raw material for this sperm whale oil, deep-sea fish orange luffy (Hoplostethus atlanticus) oil and plant jojoba (Simmondsia chinensis) oil are drawing attention. Orange Raffy oil has 16 to 22 carbon atoms,
The main component is an unsaturated ester of a long chain fatty acid and a long chain alcohol having one saturated or double bond, and jojoba oil has 20 and 22 carbon atoms, respectively.
The main component is an ester of an unsaturated long-chain fatty acid having one double bond and an unsaturated long-chain alcohol. These have a structure similar to that of sperm whale oil and can be an alternative raw material of sperm whale oil,
The former has problems with fishing restrictions and fish odors, and the latter requires a long period from sowing to harvesting.
It was not a sufficiently stable supply source.

Further, a method of producing wax ester microbiologically is known. This method is an excellent method because the growth of microorganisms is fast and the productivity can be easily adjusted.

As a conventional microbiological method for obtaining wax esters, Micrococcus cerificans is used to produce saturated wax esters such as cetyl palmitate and octadecyl palmitate from paraffins such as n-hexadecane and n-octadecane. Method (US Pat. No. 3,409,506)
No.), the carbon number is 26-30 due to the protozoa Euglena
Of producing saturated linear wax ester of
No. 9-118090), using Nocardia actinomycetes
Method for producing cetyl palmitate from hexadecane (Adv. Appl. Microbiol. 14 (1971) 93
P.), A method for obtaining unsaturated wax ester similar to sperm whale oil or jojoba oil from saturated hydrocarbon using Acinetobacter bacterium (US Pat. No. 4,404,283), Ac
Method for obtaining unsaturated wax ester by culturing inetobacter bacterium in ethanol (US Pat. No. 4,567,714)
4), etc. are known.

As a metabolic pathway for producing wax ester from n-paraffin in these microorganisms, n is the n-paraffin.
- Paraffin _(R-CH 2 -CH ₃₎ hydroxylation or by monooxygenases, by the addition of water followed by dehydrogenation to the alkene, aliphatic alcohols (R-CH ₂
Results in a -CH ₂ -OH). Further, this aliphatic alcohol is oxidized to an aliphatic aldehyde (R-CH ₂ -CHO) by the action of alcohol dehydrogenase, and further oxidized by the action of aldehyde dehydrogenase to produce a fatty acid (R
Cause -CH ₂ -COOH), and the aliphatic alcohol and the fatty acid is esterified, produce wax esters, are believed to accumulate it in the body.

Therefore, in these microorganisms, it should be possible to obtain a wax ester by assimilating an aliphatic alcohol, which is an intermediate substance of the metabolic pathway, and based on such findings, Corynebacterium sp. S-401 Method for obtaining oleyl oleate from oleyl alcohol by the method (Agric. Biol. Chem. 46 (1982) 405)
Etc. are known.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention However, of the above-mentioned known methods, all obtained by the former three methods are saturated wax esters. Although sperm whale oil is excellent as a lubricating oil as described above,
The lubricating oil must satisfy pressure resistance and aging resistance. For that purpose, the fatty acid and alcohol constituting the wax ester each have 14 or more carbon atoms, and at least one of the fatty acid and the aliphatic alcohol. It is believed necessary to have unsaturated double bonds. Therefore, the saturated wax ester as described above is not suitable as a substitute raw material for sperm whale oil.

Of the above-mentioned conventional methods, US Pat. No. 4,404,283.
According to the method described in U.S. Pat. No. 4,567,144 or US Pat. No. 4,567,144, an unsaturated wax ester is tentatively obtained, but its productivity is low and it cannot be said to be a preferable method.

Further, the method of assimilating an aliphatic alcohol to obtain a wax ester uses an expensive higher aliphatic alcohol as a raw material and is not suitable as an industrial method as well as a laboratory method.

The present invention has been made in view of the above circumstances, by assimilating fats and oils into microorganisms to decompose the fats and oils to produce fatty acids, and further reverse the metabolic pathway from the fatty acids to produce aliphatic alcohols. An object is to provide a method for producing an unsaturated wax ester from an inexpensive oil or fat in a high yield, which is produced by using the aliphatic alcohol and the fatty acid to produce an unsaturated wax ester. It is what

Means for Solving the Problems The present invention is characterized by culturing a microorganism belonging to the genus Acinetobacter, the genus Achromobacter or the genus Corynebacterium in a medium containing oil and fat.

The microorganism used in the present invention has the property of being able to assimilate n-paraffin to produce a wax ester, and specifically, it is a genus of Acinetobacter, Achromobact.
er) or a microorganism belonging to the genus Corynebacterium.

The medium and culture conditions for culturing these microorganisms include:
The medium and conditions normally used for culturing microorganisms can be used. As an example of the nitrogen sources, nitrogen sources such as ammonium chloride, ammonium sulfate, urea, ammonia, amino acids, and the like, which are assimilable to microorganisms, sodium phosphate, potassium phosphate, magnesium sulfate, manganese sulfate, ferrous sulfate, and ferric chloride are given. Iron, calcium chloride,
An inorganic salt such as manganese chloride and, if necessary, vitamins, yeast extract, and microbial growth promoting substance such as corn steep liquor are added as a basic medium.

Then, in the present invention, fats and oils are added to this medium as a carbon source, microorganisms are inoculated into the medium and aerobically cultivated at 20 to 40 ° C. while shaking, whereby the microorganisms are assimilated. . As an oil and fat that can be assimilated by microorganisms, an oil and fat containing a glycerin ester of an unsaturated fatty acid as a main component is preferable. For example, animal fats and oils such as beef tallow, lard, mink oil, vegetable fats and oils such as olive oil and rapeseed oil are suitable. At this time, promote the growth of microorganisms,
In order to smoothly produce the wax ester, acetic acid, propionic acid, fumaric acid, malic acid, lower organic acids such as pyruvic acid, glucose, fructose, hydrocarbons such as xylose, glycerin, etc., a second carbon source. It is also good to add to the medium as.

In this way, if the microorganisms utilize the fats and oils to form wax esters and accumulate them, the lipids are extracted by a method such as chloroform extraction, and the lipids are extracted from the lipids by a method such as column chromatography using silica gel. To separate the wax ester.

Action The mechanism of formation of wax ester from unsaturated fatty acid by the microorganism is considered to be as follows.

That is, the microorganism decomposes the fats and oils added to the medium to produce unsaturated fatty acids, and the unsaturated fatty acids follow the reverse pathway by the reversible reaction of the oxidation pathway of the aliphatic alcohol in the metabolic pathway of the n-paraffin. It is considered that the unsaturated fatty acid is reduced to produce an unsaturated fatty alcohol, and the unsaturated fatty alcohol and the unsaturated fatty acid are esterified to produce an unsaturated wax ester.

The wax ester synthesized through such a process is
Fatty acids as constituents of fats and oils and esters with aliphatic alcohols obtained by reducing the fatty acids are the main constituents, and by using fats and oils having unsaturated fatty acids as the main components, most of them are unsaturated fatty acids and unsaturated fatty alcohols. Becomes an ester of. The unsaturated fatty acids and unsaturated aliphatic alcohols constituting the ester mainly have the same number of carbon atoms as the fatty acid in the raw material fat and oil, and some of them have 2 or 4 less carbon atoms. .

EFFECTS OF THE INVENTION According to the present invention, unsaturated wax esters can be produced by using oils and fats that are abundantly and inexpensively supplied as a raw material, and by assimilating them into microorganisms, fatty acids in oils and fats added to the medium The unsaturated wax ester derived from the components can be efficiently produced.

Thus, the wax ester obtained by the present invention has a high proportion of unsaturated double bonds in its component, has a chemical structure similar to that of sperm whale oil, and as a substitute raw material thereof, such as a lubricant. It can be widely used for various purposes.

Examples Examples of the present invention will be described below.

NH ₄ NO ₃ 0.4g, KH ₂ PO ₄ 0.47g, NaH ₂ PO ₄・ 12H ₂
0.03 g of O, 0.05 g of MgSO ₄ .7H ₂ O and 0.5 g of powdered yeast extract were dissolved in 100 ml of tap water to adjust the pH of the solution to 7.0. A predetermined amount of a given oil or fat or an assimilating substance instead of the same was added thereto, and sterilized in an autoclave at 120 ° C. for 20 minutes to obtain a culture solution.

Then, inoculate this culture solution with the specified microorganisms, 120 times /
The cells were cultured at 28 ° C for 70 hours while shaking at min.

After that, the same amount of chloroform was added to the culture solution and shaken vigorously to extract lipids. The extracted lipid was loaded on a silica gel column and eluted with a hexane / ethyl acetate (10: 0.5) mixture to obtain a pure wax ester fraction.

The separated wax ester was saponified with an alkali, decomposed into a fatty acid component and an alcohol component, and the fatty acid component was methyl esterified, and then analyzed by gas chromatography to examine the composition of the constituent components.

Experiment 1 Acinetobacter calcoaceticus (IAM-12088) was used as a microorganism. The yields of the assimilating substance added to the culture medium and the obtained wax ester in this experiment are as follows.

1-1 assimilation substance: mink oil 1 g Yield: about 200 mg / 100 ml 1-2 assimilation substance: olive oil 1 g Yield: about 150 mg / 100 ml 1-3 Assimilation substance: rapeseed oil 1 g Yield: about 210-mg / 100 ml 1 -4 Assimilatory substance: 1 g of stearic acid Yield: Approximately 320 mg / 100 ml 1-5 Assimilable substance: 1 g of n-hexadecane Yield: Approximately 16 mg / 100 ml Experiment 2 As a microorganism, Achromobacter zerosis (Achromob)
acter xerosis) (IFO-12668) was used. The yields of the assimilating substance added to the culture medium and the obtained wax ester in this experiment are as follows.

2-1 Assimilation substance: Mink oil 1g Yield: about 190mg / 100ml 2-2 Assimilation substance: Olive oil 1g Yield: about 170mg / 100ml 2-3 Assimilation substance: Stearic acid 1g Yield: About 330mg / 100ml 2-4 Assimilating substance: Mixed n-paraffin (Nippon Oil Co., Ltd., Nisseki No. 0 Solvent M) 1 g Yield: about 47 mg / 100 ml Experiment 3 As a microorganism, Corynebacterium paurometabolum (IFO-1216)
0) was used. The yields of the assimilating substance added to the culture medium and the obtained wax ester in this experiment are as follows.

3-1 Assimilating substance: Olive oil 1 g Yield: about 110 mg / 100 ml 3-2 Assimilating substance: Nisseki No. 0 Solvent M 1 g Yield: About 10 mg / 100 ml The constituent components of the assimilating substance used in each experiment above are shown in the table. It is as 1. In the table, the numerical value in the column of "constituent component" indicates the carbon number of n-paraffin when a single numerical value is written, and in the form of N: M, N is the fatty acid component of the oil and fat. The carbon number and M indicate the number of double bonds in the fatty acid.

Table 2 shows the constituents of the wax ester obtained in each experiment. The numerical value in the column of "Constituent" in Table 2 indicates the number of carbon atoms and the number of double bonds in the fatty acid or the aliphatic alcohol as in the case of Table 1.

From the above examples, unsaturated wax esters having unsaturated fatty acids in the oil and fat added to the medium and unsaturated long-chain alcohols produced by the reduction of the unsaturated fatty acids as their constituents are efficiently produced. You can understand what you are doing.

Further, although the unsaturated wax ester is produced even when the n-paraffin is assimilated, the amount of the unsaturated wax ester produced is small, and it can be seen that the production efficiency of the unsaturated wax ester according to the present invention is extremely high.

Also, when a saturated fatty acid such as stearic acid is assimilated, a wax ester can be obtained. However, the wax ester is mainly composed of a saturated wax ester, and the content of the unsaturated wax ester is low. Not a suitable substitute for whale oil.

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Claims (1)

[Claims] 1. A method for producing an unsaturated wax ester, which comprises culturing a microorganism belonging to the genus Acinetobacter, Achromobacter or Corynebacterium in a medium containing fats and oils.