JPH01320989A - Production of wax ester - Google Patents

Abstract

PURPOSE:To efficiently mass-produce a wax ester in high purity by using a fatty acid and/or a derivative thereof as a substrate, culturing Staphylococcus lentus therein and providing a wax ester-containing lipid. CONSTITUTION:A fatty acid or a derivative thereof or both are used as a substrate and Staphylococcus lentus is cultured therein to produce a wax ester. The fatty acid or derivative or both are used as the substrate and an acyl-CoA or alcohol acyltransferase is employed as a wax ester synthetase to form a wax ester-containing lipid and produce the wax ester. As an alternative method, the fatty acid or derivative thereof or both are used as the substrate and a wax ester hydrolase obtained from Acartia clausi which is an animal plankton is used to produce the wax ester.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for producing wax esters. More specifically, we use animals, plants, and microorganisms that have the ability to synthesize wax esters, and utilize the wax ester synthase and wax ester degrading enzyme contained therein to produce oils and fats, fatty acids, higher alcohols, and higher alcohols. The present invention relates to a method for enzymatically or fermentatively producing wax esters from substrates such as dihydric alcohols.

(Prior art) Wax esters are sometimes commonly referred to as "wax" and traditionally referred to as wax, but they are synonymous with what is referred to here as wax esters. be. The wax ester here is an ester of a fatty acid and a higher-hydric alcohol or a dihydric alcohol, and is also called a fatty acid alcohol ester.

Due to their shape, wax esters can be divided into solid wax esters (e.g. carnauba wax, beeswax, etc.) and liquid wax esters (e.g. pine kola whale oil, jojoba oil, etc.).
Joba oil, etc.), and based on their source, they are classified into vegetable wax esters (e.g. carnauba wax, candelilla wax, rice wax, etc.) and animal wax esters (e.g. beeswax, wool wax, etc.) . Many wax esters are used in cosmetics, pharmaceuticals,
It is currently used in a wide range of fields including food, and has also been used for a long time as a material for wax and electrical insulators. In addition, research has been conducted on fishes from the viewpoint of their digestion and absorption, and it has been assumed that they play a specific role as feed in fish farming.

There are several reports in the United States and Japan regarding microbial methods for producing wax esters. That is, a method of obtaining wax ester as a metabolite using n-paraffin assimilating bacteria [References: S,
Dewitt et al., J^OC5, 59°69-74 (
1982) ), fermentative production method using Euglena [
References: Inui et al., Fermentation and Industry, 44.494-501
(1986); Bakusa et al., Published Patent Publication, 1986-11
8090 (1984) ), and a method for producing wax esters from unsaturated fatty acids by Acinetobacter calcoactilia [References: Kolok et al., Abstracts of the 25th Japan Oil Chemistry Symposium, p. 23 (1986), etc. It has been known.

(Problems to be Solved by the Invention) The above-mentioned wax esters derived from animal or vegetable natural bones have the drawbacks of unstable supply and inconsistent quality. Furthermore, as the whaling industry has been internationally banned in recent years, the production of animal wax esters, which have been produced using Matukora whales as raw materials, has been hit hard, and jojoba oil and its processed products are being used as alternative wax esters. Although it has come into the spotlight in countries such as the United States, it has not yet progressed beyond the research stage and is currently lacking in quantity.

Furthermore, since the wax esters obtained naturally or by fermentation contain various impurities, processes such as decolorization, deodorization, degumming, and deacidification are required. Since it requires many steps to obtain, it has the disadvantage of being too expensive. Further, it is usually difficult to obtain a wax ester having 40 or more carbon atoms.

In view of the above-mentioned problems, the present invention provides a process for efficiently producing large quantities of high-quality wax esters with high purity and in which chain length, degree of unsaturation, etc. can be arbitrarily changed. The purpose is to

(Means for Solving the Problems) The method for producing wax ester in the present invention uses a fatty acid and/or a derivative thereof as a substrate, and uses Staphylococcus 7.
This method is characterized by obtaining wax ester-containing lipids by culturing whole crabs in a supine state.

It is also characterized in that it uses a fatty acid and/or its derivative as a substrate and uses acyl-CoA:alcohol acyltransferase as a wax ester synthase.

Furthermore, fatty acids and/or their derivatives are used as substrates, and the zooplankton Acartia crucianum
It is characterized by using a wax ester degrading enzyme obtained from I.ia clausi).

The present invention will be explained in more detail below.

In the first invention, by culturing plants, animals, and microorganisms that have the ability to produce wax esters, in particular, Staphylococcus lentus, a substrate is added to the culture solution, and fermentation is performed using the microorganisms. to obtain wax ester-containing lipids.

In the second invention, wax esters are enzymatically produced using wax ester synthases found in animals, plants, and microorganisms that have wax ester production ability. Here, acyl-CoA:alcohol acyltransferase is used as the wax ester synthase.

In the third invention, a wax ester degrading enzyme in animals, plants and microorganisms having wax ester production ability, especially Acarthia crauci (Ac), which is a zooplankton.
Substrates used in the present invention include fatty acids and/or derivatives thereof, specifically fats and oils, fatty acids, higher-hydric alcohols, and higher dihydric alcohols. Examples include alcohols. In this case, fish oil having a fatty acid or alcohol having 20 or more carbon atoms can be used as the fatty acid source or the aliphatic alcohol source.

The fatty acids that are one of the substrates used in the present invention include:
All kinds of fatty acids having 2 to 24 carbon atoms can be used alone or as a mixture of two or more. Therefore, saturated fatty acids, unsaturated fatty acids, and branched fatty acids can be used. It may also be a mixture containing various fatty acids prepared from animal and vegetable oils. Particularly preferred as a fatty acid source satisfying this condition are fish oils containing fatty acids having 20 or more carbon atoms, such as sardine oil, cod liver oil, and squid liver oil.

As the higher-hydric or dihydric alcohol which is one of the substrates used in the present invention, all kinds of aliphatic alcohols having 2 to 34 carbon atoms can be used alone or as a mixture of two or more types. Therefore, regardless of whether it is saturated or unsaturated, any substance that can be used as the alcohol moiety of a wax ester, regardless of whether or not it is branched, can be used as a substrate. Alternatively, a mixture containing various aliphatic alcohols prepared from animals and plants may be used.

Moreover, it is natural that the above-mentioned fatty acid or aliphatic alcohol may be used in a free form or as a glyceride, considering the involvement of lipase.

Therefore, fats and oils such as triglycerides, partial glycerides, ether lipids, and phospholipids can also be used.

The present inventors have intensively screened microorganisms that contain a large amount of wax ester synthase, and have found that Staphylococcus lenzii has a uniquely high wax ester synthesis activity among microorganisms that have wax ester production ability.

Therefore, the present invention uses the wax ester synthase system of Staphylococcus lenz and furthermore, taking fatty acids as an example, by the reaction shown in the figure below, acyl-CoA
: Wax esters can be obtained using alcohol acyltransferase. This enzyme can be prepared from the above-mentioned plants, animals, and bacteria. +C00CHzC1, fiz□.

That is, the method for fermentative production of wax esters using microorganisms uses microorganisms with high wax ester synthesis activity, such as bacteria such as Staphylococcus lenz, and injects fatty acids, fatty alcohols, glycerides, or these into the culture solution. This is accomplished by adding a mixture.

A more detailed explanation will be given below regarding each method.

In the first invention, when culturing Staphylococcus lenz to produce wax ester-containing lipids, high concentrations of fatty acids, aliphatic alcohols, and glycerides are added to the culture medium alone or as a mixture;
I! It is preferable to use 0.1 to 150 g. These substrates can be added directly to the culture medium and stirred vigorously to form an emulsion, but in the case of fatty acids,
Preferably, the substrates may be used as water-soluble salts such as sodium or potassium salts.

Furthermore, addition of emulsifiers such as Triton X-1oo, polyvinyl alcohol, bile salts, etc., for substrate emulsification is also effective. As the carbon source, for example, glucose, fructose, sucrose, molasses, starch, wood saccharification liquid, etc. are used. It is preferable to use 60 to 400 g of carbohydrate per liter of culture medium. As the nitrogen source, for example, inorganic nitrogen sources such as ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate, etc., or organic nitrogen sources such as urea, peptone, yeast extract, corn steep liquor, etc. are used. Examples of inorganic salts include KlhPO
4, K2HPO,, NaC1, Fe5On ・7JO1
Mgso, .7H20, Zn5Oa.7H20, etc. are used. Add trace elements and other nutritional sources as necessary.

What is the usual culture of Staphylococcus lenz? This is carried out in a culture medium with aeration and agitation. The pH of the medium is 4.0
-9.0 is good<, stirring speed 300-800 r, p.

Culture is carried out for 1 to 15 days at a temperature of 1 and aeration of 10.5 to 2 m.

In this way, a wax ester-rich medium and bacterial cells can be obtained in a fermentative manner. A method for extracting total lipids rich in wax esters from the culture medium and bacterial cells is the method of Bligh & Dyer [References, E, G, Bligh & W, J, D
yer;Can, J., Brochem, P.
hysiol, + 3L 911-917 (195
Although solvent extraction methods such as those represented by 9) or supercritical carbon dioxide extraction methods are also effective, there are no particular restrictions on the extraction methods used in the present invention.

Furthermore, it goes without saying that the wax ester synthase system can be extracted and separated from the Staphylococcus lenz cells obtained in this manner, and that wax esters can be enzymatically produced as described below.

When wax ester is produced fermentatively using Staphylococcus lenz in the above method, 0.1
~150 g of wax ester enriched lipid can be obtained. Furthermore, wax ester-containing oils and fats can be continuously produced by immobilizing these microorganisms that have a high wax ester production ability, such as Staphylococcus lenzii, on a carrier.

Next, in the second invention, a case will be described in which acyl-CoA:alcohol acyltransferase is used as a wax ester synthesizing enzyme for wax ester production.

This enzyme may be used as a purified enzyme, or may be used as a crude enzyme. For batch production, the reaction solution (100
ad) The amount of enzyme added to the medium is 0.001w in terms of purified product.
A suitable amount is 3,000 mg, preferably 100 to 500 mg, but the optimum amount to be added varies depending on the types of substrates and enzyme sources, so the amount to be added is not particularly limited. The method for producing purified products is described, for example, in the references (M., AYAMA, M., MANKIJRA and
d Y, IKEDA: J, Biochem;
85. 1-6 (1979)).

In addition, the amount of fatty acid, aliphatic alcohol, or glyceride as a substrate is 10 to 15,000 mg, preferably 10
Although 0 mg to 3.000 mg can be added, the optimum amount to be added varies depending on the combination of substrates and differences in enzyme sources, so these amounts are not particularly limited. The substrates can be added as they are to the reaction solution and stirred vigorously to form an emulsion, but in the case of fatty acids, it is preferable to use the substrates as water-soluble salts such as sodium salts or potassium salts for easier handling. .

Separately, Triton X for substrate emulsification
-100, addition of polyvinyl alcohol, bile salts, sucrose fatty acid esters, and other common emulsifiers is also effective. The pH of the reaction solution is preferably in the range of 3.5 to 9.5, and it is more effective to use a buffer to adjust this pH, with a particularly preferred pH range of 5.5 to 8.0. In addition, MgZo, ATP are cofactors.
, Co1, NADH1NADI'H, etc. may also be used. The wax ester synthesis reaction is preferably carried out at a temperature in the range of 4 to 85°C; below 10°C the reaction is slow, and above 60°C generally results in deactivation of the enzyme. Therefore, 35-4
It is more preferable to carry out the reaction at 0°C. Further, although it is preferable to stir the reaction, a static reaction can also be carried out in an emulsified state. The wax ester synthesis reaction proceeds for three days or more after the start of the reaction. However, in order to prevent re-decomposition of the synthesized wax ester itself and deterioration of the enzyme protein, the reaction time is preferably within 24 hours, but considering the difference in reaction solution composition and the characteristics of the target product, the reaction time is The length is not particularly limited. The reaction may be a single-stage reaction, but may also be a multi-stage reaction that allows the reaction to proceed more efficiently. An immobilized enzyme column can also be used for continuous reaction.

When the wax ester synthesis reaction is carried out as described above, the synthesis rate usually reaches 30 to 95%.

Stopping the reaction and extracting fats and oils containing wax esters
According to the Bligh-Dyer method, it can be carried out, for example, as follows.

That is, a chloroform-methanol mixture in an amount three times the amount of the reaction solution is added, stirred vigorously for 5 minutes to mix thoroughly, and then filtered by suction. The filtrate was transferred to a separatory funnel and left overnight, then the lower layer (chloroform layer) was separated, dehydrated using anhydrous sodium sulfate or anhydrous magnesium sulfate, and the chloroform was removed under reduced pressure using an evaporator, leaving the wax ester as a residue. Oils and fats containing Alternatively, a supercritical carbon dioxide extraction method is also effective, but there are no particular restrictions depending on the extraction method.

Furthermore, the wax ester decomposing enzyme used in the third invention can be prepared from the above-mentioned plants, animals, and microorganisms that are capable of metabolizing wax esters in vivo. Such degrading enzymes can be used, for example, in the plant and animal plankton Acartia cl.
ausi). When using this enzyme, it may be used as a purified enzyme, but it may also be used as a crude enzyme.

In the case of batch production, the amount of enzyme added to the reaction solution (100d) is 0.001 to 3,000 in terms of purified product.
The appropriate amount is preferably 100 to 500 μm, but the optimum amount to be added varies depending on the enzyme source and the combination of substrates, so it is not limited to these ranges.

In addition, the fatty acids, aliphatic alcohols, or glycerides serving as substrates each have a molecular weight of 100 to 40,000, preferably 1
,000■~30,000■ can be added, but
The optimum amount to be added varies depending on the enzyme source and the combination of substrates, so it is not limited to these ranges. The substrates can be added as they are to the reaction solution and stirred vigorously to be used as an emulsion, but when fatty acids are used, it is preferable to convert the groups ff into water-soluble salts such as sodium salts or potassium salts. Easy to handle. Separately, the base f [
Addition of Triton x-too, polyvinyl alcohol, bile salts, etc. for emulsification is also effective.

In the wax ester synthesis reaction, water and an organic solvent that does not inactivate the enzyme, such as petroleum ether or hexane, are preferably used in an amount of 0.3 to 10 times, preferably 0.5 to 5 times, the amount of the substrate. Water is preferably used in a range of 0.1 to 10% based on the substrate. In the process of wax ester synthesis by this enzyme, when fatty acids and aliphatic alcohols are esterified, water production and condensation reactions are involved, so the amount of water in the system increases in proportion to the amount of wax ester synthesis. It is desirable that a dehydrating agent or the like be present in the reaction system to proceed with the reaction while dehydrating.

The reaction temperature is 10 to 60°C, preferably 30 to 40°C.
It is preferable to continue the treatment for 5 to 80 hours.

An immobilized enzyme column can also be used for continuous reaction.

That is, in order to reuse or recover the enzyme or improve reaction efficiency, it can be used by being immobilized or adsorbed on an inert support such as celite, alumina, chitosan, etc. Furthermore, the Menpuran bioreactor (Mic
roporous men+branebioreac
dehydration and wax ester synthesis reaction can be carried out simultaneously using the [References M, M, fl
og et al.: JAOC3, 61(4), 776-780
(1984)) When the wax ester synthesis reaction is carried out as described above, the synthesis rate usually reaches 20 to 90%.

Termination of the reaction and extraction of fats and oils containing wax esters can be carried out using the aforementioned Bligh-Dyer method, supercritical carbon dioxide extraction method, etc., but the difference in extraction method is not particularly restricted. Furthermore, when acyl-CoA:alcohol acyltransferase and wax ester degrading enzyme coexist as wax ester synthases, they can be treated in the same manner as described above.

(Effects of the Invention) According to the method of the present invention, using Staphylococcus lenz, or using acyl-CoA:alcohol acyltransferase, or using a wax ester degrading enzyme obtained from the zooplankton Acarthia crucii, Wax esters can be produced enzymatically or fermentatively in large quantities with high yield and high purity.

These products can be used for food, cosmetics, medicine, etc. For example, if wax ester production is performed by the method of the present invention using valmitic acid and/or oleyl alcohol as a substrate, oleyl palmitate, which is one of the main components of whale oil, can be efficiently produced.

(Examples) Hereinafter, the present invention will be specifically explained based on Examples, but the present invention is not limited to these Examples.

Example 1 Staphylococcus lentsun
An example of a method for producing oils and fats containing wax esters by cultivating .tus) will be described.

As a medium, add 30 g of dry broth to 1
After adding it to the purified water of step 1 and dissolving it by heating, it was dispensed into a predetermined container and sterilized in high-pressure steam at 121° C. for 15 minutes. Table 1 shows the main component composition and pH.

Table 1 Medium II! Composition inside (pH 7.0 ± 0.1) (unit: g) Meat extract 5.0 peptone
15.0 Sodium chloride
5.0 Potassium hydrogen phosphate 5.0 total
30.0 In the medium shown in Table 1,
Triton
, 0g) and added to the medium, and then inoculated with Staphylococcus lenz. Needless to say, the above medium did not contain wax ester. 40℃
After culturing for one week, the culture solution containing the bacterial cells was
When total lipids were collected using the diamond method, the content was 2.
It was 0.5g. In addition, no contamination with bacteria was observed during the culture period.

When the obtained total lipids were qualitatively examined by thin layer chromatography, wax esters, free fatty acids, free aliphatic alcohols, as well as phospholipids, free sterols, triazylglycerol, etc. were confirmed. 0.5 g of the total lipid was fractionated by silicic acid column chromatography to obtain 0.13 g of wax ester fraction. Therefore, 5.33 g of wax ester (
(mainly as oleyl oleate) was produced.

Example 2 An example of a wax ester production method using acyl-CoA:alcohol acyltransferase using a fatty acid as a substrate is shown.

Staphylococcus nocuslens cells obtained by culturing for one week at 40°C in the dry bouillon medium shown in Example 1 were disrupted by a conventional method, and centrifuged (10,000x
g, 10 minutes), the supernatant was fractionated with ammonium sulfate in 0.01 M phosphate buffer (pH 7.0), and the O~50% fraction (0.
After dialysis, this was used as a crude enzyme preparation.

Linoleic acid (1.0 g) as a substrate was added to the potassium salt reaction solution. In addition, ATP, C as cofactors
oA, NADHSNADPII, etc. were added in the proportions shown in Table 2, and the volume of the reaction solution was finally adjusted to 100% with 0.01 M phosphate buffer (pH 7,0), and the mixture was heated at 35°C for 2 hours.
Incubation was for 4 hours.

*The total volume was adjusted to 100- with 0.01M phosphate buffer (pH 7.0).

After the reaction was completed, all lipids were extracted from each section according to the Bligh-Diaper method, and the Fuchs ester content was determined in the same manner as in Example 1. No. 1 (0.11 g), No. 2
(0,37g), No, 3 (0,25g), No,
4 (0.35g). That is, in the case of this example, in order to produce wax ester from fatty acid using acyl-CoA:alcohol acyltransferase,
MgZ+, TP, NADII, or N was used to activate the enzyme system that reducts fatty acids to aliphatic alcohols contained in the crude enzyme preparation used to obtain the fatty acid counterpart compound.
The presence of ADPI+ is effective. Further, CoA is effective in synthesizing CoA FA conductor, which is an intermediate for wax ester synthesis.

Example 3 An example of a wax ester production method using an aliphatic alcohol as a substrate using acyl-CoA:alcohol acyltransferase is shown.

Fresh carp liver pancreas III (50g) was obtained from a carp fish farm (4 carp fishes, anus, and gods), and it was heated to 2x Wk under water cooling.
O) Homogenized with 0,01M phosphate buffer (pH 7,0) and centrifuged (10,000 x g
, 10 minutes), the supernatant was fractionated with ammonium sulfate to obtain a 0-50% fraction (1.0 g), which was used as an enzyme preparation after dialysis.

As a substrate, oleyl alcohol (2.0 g) was emulsified with Triton X-100 (0.2 g) and added to the reaction solution. ATP and CoA were added as cofactors as shown in Table 3. Finally, the volume of the reaction solution was reduced to 100% by adding 0.01M phosphate buffer (pH 7.0), and 30%
Incubated at ℃ for 24 hours.

*The total volume was adjusted to 100- with 0.01M phosphate buffer (pH 7.0).

After the reaction was completed, all lipids were extracted from each section by the Bligh-Dia method, and the Fuchs ester content was determined in the same manner as in Example 1. No. 1 (0.32 g), No. 2
(1,28g) and No.3 (1,63g). Therefore, it can be seen that addition of ATP, CoA, etc. to the reaction solution is more effective.

Example 4 An example of a method for producing wax ester from fatty acid or aliphatic alcohol using a wax ester degrading enzyme is shown.

Obtain 100 g (as wet weight) of Acartia clausis, a zooplankton rich in wax esters, and add twice the volume of 0.01 M phosphate buffer (pH 7.0) under water cooling.
Homogenize and centrifuge (10,000x
g, 10 min), the supernatant was fractionated with ammonium sulfate and
% fraction was obtained (1.0 g). Sephadex (S) after dialysis
ephadex) G-200 column to obtain a crude product of the wax ester degrading enzyme fraction (
Protein content 120mg10. OIM phosphate buffer (
pH 7,0) 9-). Oleic acid and oleyl alcohol were added as substrates together with n-hexane, and the mixture was reacted at 30° C. for 48 hours with vigorous stirring using a magnetocooler. Table 4 shows the reaction solution composition.

Table 4 * Crude enzyme preparation (contains 120 μg of protein) is 9 m
1 of 0.001M phosphate buffer (pl+7.0).

After the reaction was completed, the mixture was dehydrated by centrifugation (6,000×g, 30 minutes) to recover oil and fat components, and the wax ester content was determined in the same manner as in Example 1. No. 1 (21
g, synthesis rate 10.5%), No. 2 (88 g, synthesis rate 22%), No. 3 (78 g 1 synthesis rate 13.0
g), No. 4 (69 g, synthesis rate 8.6%).

Example 5 An example of a method for culturing Staphylococcus lenzi and producing fats and oils containing a large amount of wax ester from triglycerides will be described.

50 g refined sardine oil as substrate () Liglyceride content 9
9% or more), and the medium composition in Table 5 was used.

Table 5 Composition of 1 liter of culture medium (pH 7,5) Dilute to 11 with ion-exchanged water.

After culturing at 35°C for 4 days, total lipids were recovered from the culture solution containing the bacterial cells by the Bligh-Dyer method, and the content was 41 g. Purification and fractionation was performed according to the method of Example 1, and 18.0 g of wax ester was obtained. Therefore, 18.0 g of wax was produced per 11 medium, giving a yield of 36.0% based on the amount of substrate provided.
becomes. At this time, when the chain length distribution of the wax ester obtained was determined by GLC, a distribution as shown in Table 6 was obtained.

Table 6 Analytical conditions were conducted according to the literature. (Citation: M,
KAYAMA et al., Yukagaku, 2
3. 290-295 (1974)).

From this table, it can be seen that most of the wax esters have carbon atoms of 30 or more, and that about 20% of the total wax esters have particularly high physiological activity and have carbon atoms of 40 or more.

Claims (3)

[Claims] (1) Using fatty acids and/or derivatives thereof as a substrate,
A method for producing wax esters, which comprises obtaining wax ester-containing lipids by culturing S. lentus. (2) A method for producing wax esters, characterized in that a fatty acid and/or a derivative thereof is used as a substrate and acyl-CoA:alcohol acyltransferase is used as a wax ester synthase. (3) Using fatty acids and/or their derivatives as a substrate, the zooplankton
1. A method for producing wax esters, which comprises using a wax ester degrading enzyme obtained from A. aclausi.