JP7649371B2 - Method for producing ketooctadecadienoic acid - Google Patents

Description

The present invention relates to a method for producing ketooctadecadienoic acid.

In recent years, hydroxylated fatty acids and oxo fatty acids obtained by reacting linoleic acid or oleic acid with enzymes or other means have been attracting attention as so-called rare fatty acids. Along with growing interest in people's health and medicine, these rare fatty acids are expected to be applied to various industrial uses, particularly due to their physiological activity. In particular, 13-oxo-octadecadienoic acid and 9-oxo-octadecadienoic acid, which are types of ketooctadecadienoic acid, which is an oxo derivative of linoleic acid, have been found to have activity in improving lifestyle-related diseases such as improving lipid metabolism, and are therefore being actively researched both domestically and internationally as functional ingredients that show a remarkable fat-burning effect.

Ketooctadecadienoic acid has long been known to be contained in tomatoes, but the amount is so small that it is only about 1 ng/mg, and is therefore not a sufficient source for use as an active ingredient in foods or medicines. Therefore, there has been a demand for a source containing ketooctadecadienoic acid at a higher content, and for a more efficient synthesis of ketooctadienoic acid.

For example, Patent Document 1 describes a method for synthesizing ketooctadecadienoic acid, which includes a step of reacting an enzyme material containing lipoxygenase with a raw material containing linoleic acid in the presence of a metal such as manganese, zinc, iron, copper, or magnesium at a temperature of less than 20°C.

JP 2016-178913 A

The method for producing ketooctadecadienoic acid described in Patent Document 1 uses a metal as a catalyst, and when the synthesized ketooctadecadienoic acid is used for food or pharmaceutical purposes, there is a risk that the product will contain metal. In addition, in order to suppress the production of by-products and improve the yield, it is essential to keep the reaction temperature below 20°C in the method for producing ketooctadecadienoic acid described in Patent Document 1, and the production process requires complicated equipment and machinery for temperature adjustment in the production facilities.

In addition, in the method for producing ketooctadecadienoic acid described in Patent Document 1, lipoxygenase is used as an enzyme. The addition of dehydrogenase together with lipoxygenase as an enzyme to be used is also suggested, but since lipoxygenase itself is an enzyme that significantly promotes the reaction of fatty acids, a large amount of peroxide is generated by lipoxygenase, particularly at reaction temperatures of 20°C or higher, which further oxidizes the generated ketooctadecanoic acid. This causes a problem in that the final yield of the target substance, ketooctadecanoic acid, is reduced. There is a demand for a method for producing ketooctadecadienoic acid that uses enzymes to synthesize ketooctadecadienoic acid safely, stably, and efficiently.

The present invention aims to provide a method for producing ketooctadecadienoic acid, which has been difficult to supply until now, using enzymes to safely, stably, and efficiently produce the acid.

The present invention relates to a method for producing ketooctadecadienoic acid, comprising the steps of (a) reacting an enzyme material containing lipoxygenase with a raw material containing fatty acids, and (b) terminating the enzymatic reaction of the lipoxygenase contained in the reaction mixture obtained in step (a).

The present invention further relates to a method for producing ketooctadecadienoic acid, which includes (c) a step of reacting the product obtained through the step (b) with a dehydrogenase.

The fatty acid-containing raw material is preferably a raw material containing an oil or fat containing linoleic acid as a constituent, a raw material containing linoleic acid, or a method for producing ketooctadecadienoic acid, which is linoleic acid.

The method for producing ketooctadecadienoic acid is preferably one in which the step of reacting the enzyme material is carried out at a temperature of 10°C or higher and 60°C or lower.

The method for producing ketooctadecadienoic acid is preferably one in which the step of reacting the enzyme material is carried out at a temperature of 20°C or higher and 50°C or lower.

In the process of allowing the enzyme material to act, the method for producing ketooctadecadienoic acid is preferably one in which the enzyme material is allowed to act for 0.5 hours or more and 72 hours or less.

The method for producing ketooctadecadienoic acid is preferably one in which the step of terminating the enzyme reaction is carried out by inactivating the enzyme activity of the lipoxygenase.

Preferably, the method for producing ketooctadecadienoic acid involves deactivating the acid by heat treatment or pH adjustment.

The heat treatment is preferably carried out by heating the reaction mixture to 70°C or higher and 140°C or lower in the method for producing ketooctadecadienoic acid.

Preferably, the method for producing ketooctadecadienoic acid involves adjusting the pH of the reaction mixture to less than pH 6 or more than pH 12.

The method for producing ketooctadecadienoic acid is preferably one in which the step of terminating the enzyme reaction is carried out by removing the lipoxygenase from the reaction mixture.

In the method for producing ketooctadecadienoic acid, the step of terminating the enzyme reaction is preferably carried out by adding an inhibitor of the lipoxygenase to the reaction mixture to inhibit the enzyme activity of the lipoxygenase.

The method for producing ketooctadecadienoic acid is preferably such that the dehydrogenase is an alcohol dehydrogenase.

The method for producing ketooctadecadienoic acid is preferably one in which the dehydrogenase is allowed to act in the presence of a cofactor.

A method for producing ketooctadecadienoic acid in which the cofactor is NAD ⁺ is preferred.

The method for producing ketooctadecadienoic acid is preferably one in which the dehydrogenase reaction step is carried out at a temperature of 0°C or higher and 50°C or lower.

The method for producing ketooctadecadienoic acid is preferably one in which the dehydrogenase action step is carried out at a pH of 6 or more and 12 or less.

A preferred method for producing ketooctadecadienoic acid involves allowing the enzyme material containing lipoxygenase to act on a raw material containing the fatty acid, and then subjecting the raw material to the heat treatment within 0.5 to 24 hours.

According to the method for producing ketooctadecadienoic acid of the present invention, two different enzymes can be reacted independently and sequentially, so that the amount of by-products produced, which was a problem in terms of reaction yield, can be significantly reduced. Therefore, ketooctadecadienoic acid can be produced efficiently. In addition, the synthesis temperature can be room temperature, and ketooctadecadienoic acid can be produced with sufficient yield even at a reaction temperature of 20°C or higher. Furthermore, the method for producing ketooctadecadienoic acid of the present invention does not require the use of special metals. Since only two types of enzymes are used, ketooctadecadienoic acid can be produced by a simpler method than conventional techniques.

Ketooctadecadienoic acid is one of the metabolites obtained by the oxidative metabolism of linoleic acid in certain microorganisms and plants. Linoleic acid is enzymatically converted to lipid peroxide (hydroperoxyoctadecadienoic acid (HPODE)) by the action of lipoxygenase (LOX), a linoleic acid metabolic enzyme, and this lipid peroxide (HPODE) is converted non-enzymatically or enzymatically, for example by peroxidase, to hydroxyoctadecadienoic acid (HODE), a hydroxylated fatty acid, by releasing radicals. Ketooctadecadienoic acid, an oxo derivative of fatty acid, is produced by the action of dehydrogenases such as alcohol dehydrogenase (ADH) on hydroxyoctadecadienoic acid (HODE).

Specific examples of ketooctadecadienoic acids include 9-oxo-10,12-octadecadienoic acid (also abbreviated as 9-oxoODA in this specification), 13-oxo-9,11-octadecadienoic acid (also abbreviated as 13-oxoODA in this specification), 5-oxo-6,8-octadecadienoic acid, 6-oxo-9,12-octadecadienoic acid, 8-oxo-9,12-octadecadienoic acid, 10-oxo-8,12-octadecadienoic acid, 11-oxo-9,12-octadecadienoic acid, 12-oxo-9,13-octadecadienoic acid, and 14-oxo-9,12-octadecadienoic acid. In addition, when isomers exist in these compounds, all isomers and mixtures thereof are included.

The present invention provides a method for producing ketooctadecadienoic acid with high efficiency by enzymatically converting linoleic acid into hydroperoxyoctadecadienoic acid (HPODE) or hydroxyoctadecadienoic acid (HODE) using lipoxygenase (LOX), inactivating LOX, and then acting on the resulting mixture with a dehydrogenase such as ADH as necessary. LOX may be inactivated after HPODE or HODE is produced. The following describes in detail the embodiments for carrying out the present invention. However, the present invention is not limited to the following embodiments.

In some embodiments of the present invention, ketooctadecadienoic acid is produced from a raw material containing a fatty acid. Examples of the raw material containing a fatty acid include a raw material containing an oil and fat containing a fatty acid as a component, a raw material containing a fatty acid, and a fatty acid. Preferably, the raw material containing a fatty acid is a raw material containing an oil and fat containing linoleic acid as a component, a raw material containing linoleic acid, or linoleic acid. Here, linoleic acid is not limited to linoleic acid, and may be conjugated linoleic acid or may include an ester of linoleic acid.

The raw material containing fats and oils containing linoleic acid as a component or the raw material containing linoleic acid is not particularly limited, and for example, any raw material containing fats and oils containing linoleic acid as a component in a plant, animal, or bacterial body or a raw material containing linoleic acid can be suitably used in the manufacturing method of this embodiment. If a plant body such as soybean, castor, safflower, or corn, which is known to have a high content of fats and oils containing linoleic acid as a component or linoleic acid, or an animal body such as eggs is used, it may be preferable to use such a raw material, since it is possible to produce ketooctadecadienoic acid in high yield. In addition, high-purity linoleic acid extracted and/or purified from the above-mentioned raw material may be used as the raw material.

In some embodiments of the present invention, ketooctadecadienoic acid is produced by reacting an enzyme material containing lipoxygenase with a raw material containing the above-mentioned fatty acid. As the enzyme material containing lipoxygenase, a plant, animal or bacterial material containing lipoxygenase may be used. For example, the lipoxygenase contained in the above-mentioned raw material may be used as it is as a naturally occurring lipoxygenase contained in the enzyme material. From the viewpoint of efficiently promoting the oxidation reaction of fatty acids, a naturally occurring lipoxygenase, i.e., a plant-derived, animal-derived or bacterial-derived lipoxygenase purified by an appropriate isolation and purification technique from a plant body such as soybean, an animal body such as egg, or a bacterial body may be used. Furthermore, a lipoxygenase produced as a recombinant by genetic engineering techniques or a chemically synthesized lipoxygenase may be used. Furthermore, a commercially available product may be used. Furthermore, the enzyme material may contain other enzymes in addition to lipoxygenase.

The amount of enzyme material used can be selected appropriately depending on the raw material used. For example, an enzyme material containing 100 to 300,000 units of lipoxygenase in purified enzyme equivalent per 10 g of linoleic acid contained in the raw material is used. 2,000 to 180,000 units of lipoxygenase may be used per 10 g of linoleic acid contained in the raw material. Also, 5,000 to 90,000 units of lipoxygenase may be used. In this specification, one unit is defined as the amount of enzyme that increases the absorbance of a substrate solution at a wavelength of 234 nm by 0.001 in one minute at pH 9 and temperature 25°C in a 3 mL (1 cm wide) quartz cell using linoleic acid as a substrate. Therefore, one unit of lipoxygenase activity here corresponds to the amount of oxidizing 0.12 μmol of linoleic acid per minute.

The reaction temperature at which the enzyme material is allowed to act on the raw material containing fatty acids can be set appropriately depending on the raw material used, but is preferably 0 to 90°C. Below 0°C, the solvent may freeze and the reaction may not proceed. At temperatures higher than 90°C, the LOX contained in the enzyme material is quickly deactivated. For example, the reaction temperature at which the enzyme material is allowed to act can be 10°C or higher and 60°C or lower. Preferably, the reaction temperature at which the enzyme material is allowed to act can be 15°C or higher and 50°C or lower. Preferably, the reaction temperature can be 20°C or higher and 50°C or lower. Preferably, the reaction temperature is 30°C or higher and 50°C or lower.

The reactions or steps described herein can be carried out in a suitable solvent. Suitable solvents can be readily selected by one of ordinary skill in the art. A suitable solvent is one that is substantially non-reactive with the reactants, intermediates and/or products of the reactions or steps described herein at the temperatures at which the reactions are carried out. The reactions or steps described herein can be carried out in one solvent or in a mixture of two or more solvents.

The time for which the enzyme material is allowed to act on the raw material containing fatty acids may be set appropriately depending on the LOX content in the enzyme material and the reaction temperature, but for example, 0.5 to 144 hours is preferable. If the time for acting is shorter than 0.5 hours, there is a risk that the oxidation reaction by LOX will not proceed. If the time for acting the enzyme material is longer than 72 hours, side reactions are more likely to occur. It is more preferable that the time for acting the enzyme material is approximately 0.5 to 72 hours. Furthermore, it is more preferable that it is 24 to 72 hours.

The reaction in which the enzyme material containing LOX acts can be carried out in a pH range of about 6 to 12. At a pH below 6 or above 12, the LOX contained in the enzyme material may be inactivated and/or denatured in the reaction solution. In particular, the reaction in which the enzyme material containing LOX acts is preferably carried out under conditions of pH 9 to 12. Furthermore, it is preferable to inactivate LOX under conditions of pH 10 to 12. At a pH within this range, the linoleic acid contained in the raw material may be easily emulsified, and the reaction may proceed easily. The pH in the reaction can be adjusted using a known pH adjuster, for example, potassium salts such as potassium hydroxide, potassium carbonate, potassium hydrogen carbonate, or potassium hydrogen phosphate, sodium salts such as sodium hydroxide, sodium carbonate, sodium hydrogen carbonate, or sodium hydrogen phosphate, amines such as triethylamine, or ammonia.

By the reaction between the raw material containing the fatty acid and the enzyme material containing lipoxygenase, oxygen molecules are added to the fatty acid to generate lipid peroxides and/or hydroxylated fatty acids. In the method for producing ketooctadecadienoic acid of the present invention, the enzyme reaction of lipoxygenase is then stopped in the reaction mixture obtained by the reaction between the raw material containing the fatty acid and the enzyme material containing lipoxygenase.

The enzyme reaction of lipoxygenase can be stopped, for example, by inactivating the enzyme activity of lipoxygenase, although there is no particular limitation. Examples of methods for inactivating the enzyme activity of lipoxygenase include inactivation of lipoxygenase activity by heat treatment and inactivation of lipoxygenase activity by pH adjustment. The enzyme reaction of lipoxygenase can also be stopped by removing lipoxygenase from the reaction mixture, for example, by ultrafiltration or gel filtration, or by passing the reaction mixture through a reaction column such as a column using a so-called immobilized enzyme in which the enzyme is adsorbed or bound to the surface of a carrier or is encapsulated in a gel. Alternatively, the enzyme reaction of lipoxygenase can be stopped by adding a reagent such as an inhibitor that interacts with lipoxygenase to selectively inhibit the reaction of lipoxygenase to the reaction mixture. That is, in the broad sense of the present specification, stopping the enzyme reaction of lipoxygenase includes not only inactivating the enzyme activity of lipoxygenase, but also inhibiting the enzyme activity of lipoxygenase and removing lipoxygenase.

In some embodiments of the present invention, the enzyme reaction of lipoxygenase can be stopped by inactivating the lipoxygenase activity by heat-treating the reaction mixture. Inactivation by heat treatment is particularly preferred because it can be easily controlled and can be efficiently performed even in large-scale production. The heating temperature and holding time of the heat treatment can be appropriately set to conditions that allow lipoxygenase to be sufficiently inactivated. For example, the heating temperature is preferably 70°C or higher. When it is preferable to inactivate the enzyme activity of lipoxygenase in a shorter time, it is preferable to set the heating temperature to 90°C or higher. There is no particular upper limit for the heating temperature, but a temperature at which the solvent in the reaction mixture does not volatilize too much is preferred, and for example, it can be about the temperature (120 to 140°C) used in general autoclave treatment. By performing the step of stopping the enzyme reaction of lipoxygenase at this temperature, for example, when the produced ketooctadecadienoic acid is used for food or pharmaceutical purposes, the product is heat-sterilized at the same time as the lipoxygenase activity is inactivated, which may be particularly preferred. The retention time, i.e., the time for inactivating lipoxygenase, is not particularly specified, but it can generally be 5 minutes or more, and preferably 10 minutes or more.

When the inactivation of lipoxygenase activity is carried out by pH adjustment, the pH of the reaction mixture is adjusted to less than pH 6 or greater than pH 12. The pH adjustment can be carried out using known pH adjusters as described above.

In some embodiments of the present invention, the enzymatic reaction of lipoxygenase is stopped by inhibiting the enzymatic activity of lipoxygenase by adding a reagent, such as an inhibitor, to the reaction mixture. Lipoxygenase inhibitors include, for example, N-hydroxyurea derivatives, redox inhibitors, and non-redox inhibitors, including, but not limited to, N-[1-benzo[b]thien-2-ylethyl]-N-hydroxyurea (zileuton), 1-[4-[5-(4-fluorophenoxy)-2-furyl]but-3-yn-2-yl]-1-hydroxy-urea, tetrahydro-4-[3-[[4-(2- Examples include methyl-1H-imidazol-1-yl)phenyl]thio]phenyl)-2H-pyran-4-carboxamide (CJ-13610), 2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-cyclohexa-2,5-diene-1,4-dione (docebenone), and 6-[[3-fluoro-5-(4-methoxyoxan-4-yl)phenoxy]methyl]-1-methyl-quinolin-2-one (ZD2138).

In some embodiments of the present invention, the enzymatic reaction of lipoxygenase is subsequently stopped, and then a dehydrogenase is applied to the resulting product to carry out a dehydrogenation reaction. This produces ketooctadecadienoic acid. The dehydrogenase used here is not particularly limited as long as it is an enzyme that can use the above-mentioned lipid peroxides or hydroxylated fatty acids as substrates and convert them into oxo derivatives of fatty acids, but is preferably alcohol dehydrogenase (ADH). The origin of the enzyme is also not particularly limited. ADH derived from plants such as cucumber or ADH derived from microorganisms such as yeast may be used, and ADH derived from animals may also be used. ADH produced as a recombinant by genetic engineering techniques or ADH chemically synthesized may also be used. Furthermore, commercially available ADH can also be used. In order to proceed with the reaction more efficiently, ADH purified from plants or yeast can be used.

In some embodiments of the present invention, the step of allowing the dehydrogenase to act is preferably carried out in the presence of a cofactor. The cofactor may be added to the product obtained through the step of stopping the enzymatic reaction of lipoxygenase. The cofactor may also be added to the product as a mixture with the enzyme. The cofactor may be selected depending on the type of dehydrogenase. Examples of the cofactor may include, but are not limited to, nicotinamide adenine dinucleotide (NAD ⁺ ), nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and the like. Preferably, the cofactor is nicotinamide adenine dinucleotide (NAD ⁺ ). The concentration of the cofactor added may be such that the dehydrogenation reaction proceeds efficiently.

The amount of ADH used can be appropriately selected depending on the raw material used. For example, 10 to 15,000 units of ADH, calculated as purified enzyme, may be used for 10 g of linoleic acid contained in the raw material. More than 15,000 units of ADH may also be used. 20 to 5,000 units of ADH may also be used, or 100 to 500 units of ADH may also be used. In this specification, 1 unit is defined as the amount of enzyme that converts 1.0 μmol of ethanol to acetaldehyde in 1 minute under conditions of pH 8.8 and 25°C.

The temperature at which ADH is allowed to act can be set appropriately depending on the raw materials used, but is preferably between 0 and 50°C. If the temperature is below 0°C, the solvent may freeze and the reaction may not proceed. Furthermore, if the temperature is higher than 50°C, the ADH enzyme will be quickly inactivated. More preferably, the temperature may be between 10°C and 40°C.

The time for which ADH is allowed to act can be set appropriately depending on the reaction temperature, etc., but is preferably about 1 to 72 hours. From the standpoint of reaction efficiency, an ADH action time of about 24 hours is sufficient.

The reaction in which ADH acts is preferably carried out within a pH range of about 6 to 12. At a pH below 6 or above 12, ADH may be inactivated and/or denatured. In particular, it is preferable to carry out the reaction under conditions of pH 8 to 12. Therefore, the pH conditions can be similar to those in the step in which the enzyme material containing LOX is acted on, and in this case, it is preferable because there is no need to adjust the pH again in the step in which ADH acts on. The pH in the reaction can be adjusted using a known pH adjuster, for example, potassium salts such as potassium hydroxide, potassium carbonate, potassium hydrogen carbonate, or potassium hydrogen phosphate, sodium salts such as sodium hydroxide, sodium carbonate, sodium hydrogen carbonate, or sodium hydrogen phosphate, amines such as triethylamine, or ammonia.

By applying ADH to the product obtained after the step in which the enzyme reaction of lipoxygenase is stopped, the lipid peroxides and fatty acid hydroxides, which are lipoxygenase metabolites of fatty acids that were previously produced by the reaction with lipoxygenase, are converted to ketooctadecadienoic acid. Therefore, according to the production method of this embodiment, the production of by-products is significantly suppressed, and ketooctadecadienoic acid can be stably produced.

In an embodiment of the present invention, after HPODE is produced, LOX is inactivated by heat treatment, and ketooctadecadienoic acid can be obtained by subsequent heat treatment. In this case, the enzyme material containing lipoxygenase is allowed to act on the raw material containing fatty acids, and then heat treatment is carried out within 0.5 to 24 hours, with the heat treatment time being 10 minutes or more, preferably 30 minutes or more.

The steps described herein can be monitored according to suitable analytical methods known in the art. The production of the products can be monitored by various spectroscopic means or by chromatography. According to the production method of the present invention, among ketooctadecadienoic acids, 9-oxo-10,12-octadecadienoic acid (9-oxoODA) and 13-oxo-9,11-octadecadienoic acid (13-oxoODA) are produced from a raw material containing linoleic acid. The production of 9-oxoODA, 13-oxoODA, etc. can be confirmed by analytical means such as LC-MS, and the concentration can also be quantitatively analyzed. Thus, the conversion yield of the product from linoleic acid, so-called yield, can be calculated.

As mentioned above, ketooctadecadienoic acid has various isomers, such as (E,E), (Z,E), (E,Z), and (Z,Z), but their effects as functional ingredients are roughly equivalent. Therefore, the yield of ketooctadecadienoic acid described in the following examples is the combined yield of these isomers.

The method for producing ketooctadecadienoic acid according to the above-mentioned embodiment may additionally include a step of purifying the produced ketooctadecadienoic acid. For example, the produced ketooctadecadienoic acid may be separated and purified by a general purification method, for example, by thin layer chromatography or liquid chromatography after solvent extraction. The purification method and conditions may vary depending on the experiment, and for example, the eluent, solvent, stationary phase, etc. in the chromatography and HPLC purification may be appropriately selected. However, the produced ketooctadecadienoic acid may be used as it is without purification, as a reaction solution containing ketooctadecadienoic acid, or as a crude product that has been subjected to a crude purification process, for example, dried after solvent extraction.

Among the ketooctadecadienoic acids produced by the method for producing ketooctadecadienoic acid according to the embodiment described above, the 9-oxoODA form and the 13-oxoODA form have been reported to activate PPARα, and are expected to have fat burning effects (for example, Kim YI, et al., Mol. Nutr. Food. Res., 55 (2011) 585. and Young-il Kim, et al., PLoSONE, 7 (2012) 2), and can be used to prevent and improve, for example, obesity, lipid metabolism disorders, diabetes caused by insulin resistance, hyperlipidemia, arteriosclerosis, and heart disease. For example, for practical use, they can be added to supplements, foods, functional foods, cosmetics, etc.

The inventors have also discovered that ketooctadecadienoic acid can be used as a plant activator that exhibits a strong resistance-inducing effect when applied to plants, and have found that it exhibits growth-promoting effects in various plants, fruit yield-increasing effects, and disease-suppressing effects. Specific examples of disease suppression include gray mold, vine splitting, vine wilt, and downy mildew on the leaves of Cucurbitaceae plants such as cucumbers, watermelons, melons, and pumpkins; bacterial wilt, wilt, half-leaf wilt, damping-off, and brown root rot on Solanaceae plants such as tomatoes, eggplants, and potatoes; powdery mildew, black spot, gray mold, and anthracnose on Rosaceae plants such as roses and strawberries; downy mildew on Amaranthaceae plants such as spinach; black rot, soft rot, bacterial spot disease, Rhizoctonia disease on Brassica plants such as Chinese cabbage, cabbage, and Komatsuna; southern blight on Apiaceae plants such as carrots; and rice blast on Gramineae plants.

According to the method for producing ketooctadecadienoic acid according to the embodiment described above, ketooctadecadienoic acid can be produced simply, stably, and efficiently using two different enzymes. Since ketooctadecadienoic acid, which has been difficult to supply until now, can be produced with a sufficient yield, it is expected that ketooctadecadienoic acid will be applied to physiological activity analysis and industrial use. Furthermore, since ketooctadecadienoic acid can be stably produced without the need for the addition of metals, the method for producing ketooctadecadienoic acid of the present invention is also useful in that the produced ketooctadecadienoic acid can be safely used in various fields, and the obtained ketooctadecadienoic acid is particularly suitable as a raw material for functional foods, pharmaceuticals, cosmetics, plant activators, etc.

In addition, according to the method for producing ketooctadecadienoic acid according to the embodiment described above, ketooctadecadienoic acid can be produced efficiently at room temperature by reacting two different enzymes independently and sequentially, and since no special temperature control device or temperature control is required, it is believed that labor, resource, and energy saving in production, and ultimately reduction in production costs, can be achieved.

The present invention will be described based on examples, but the present invention is not limited to only the examples.

Preparation of Ketooctadecadienoic Acid - Example 1
As a raw material containing fatty acids, 2.8 g of linoleic acid (manufactured by Wako Pure Chemical Industries, Ltd.) with a purity of 80% was used, and 7.0 g of potassium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) and 300 mL of distilled water were added thereto to prepare a test solution. The pH of the test solution at this time was 11.

0.2 mg of lipoxygenase (Sigma-Aldrich, derived from Glycine max) was added to the test solution and reacted at 30°C for 36 hours, after which the reaction mixture was placed in a 90°C water bath for 5 minutes to inactivate the enzyme. After the reaction solution was returned to room temperature, 0.2 mg of alcohol dehydrogenase (Wako Pure Chemical Industries, Ltd., derived from yeast) was added and reacted at 30°C for an additional 24 hours.

After the reaction, the products in the test solutions were identified by LC-MS using MS ² spectrum analysis with 13-oxoODA (Cayman Chemical) as a standard substance. Quantitation was performed by the absolute calibration curve method at a detection wavelength of UV 272 nm. 9-oxoODA was quantified with reference to the absorbance of 13-oxoODA.

As shown in Table 1, 13-oxoODA was obtained in a yield of 5.4% as a combined yield of isomers such as (E,E isomer) and (E,Z isomer). The yield of 9-oxoODA was 10.9%. The yield (%) was calculated based on the following formula.
Yield (%)=(wt% of 13-oxoODA or 9-oxoODA produced)/(initial wt% of raw linoleic acid used)

Example 2
The same procedures and analyses were carried out as in Example 1, except that the reaction time of the test solution with lipoxygenase was changed from 36 hours to 24 hours.

As shown in Table 1, the combined yield of isomers such as (E,E isomer) and (E,Z isomer) was 3.5% for 13-oxoODA. The yield of 9-oxoODA was 3.7%.

Example 3
As a raw material containing fatty acids, 2.8 g of linoleic acid (manufactured by Wako Pure Chemical Industries, Ltd.) with a purity of 80% was used, and 4.0 g of potassium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.), 23.3 g of dipotassium hydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.), and 300 mL of distilled water were added to prepare a test solution. The pH of the test solution at this time was 9.0.

0.4 mg of lipoxygenase (Sigma-Aldrich, derived from Glycine max) was added to the test solution and reacted at 15°C for 3 hours. The reaction mixture was then placed in a 90°C water bath for 90 minutes to inactivate the enzyme. The product after the reaction was treated and analyzed in the same manner as in Example 1.

As shown in Table 1, the combined yield of isomers such as (E,E isomer) and (E,Z isomer) was 2.6% for 13-oxoODA. The yield of 9-oxoODA was 1.2%.

Comparative Example 1
As a raw material containing fatty acids, 0.1 g of linoleic acid with a purity of 80% (manufactured by Wako Pure Chemical Industries, Ltd.) was used, and 0.23 g of potassium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) and 10 mL of distilled water were added thereto to prepare a test solution. The pH of the solution at this time was 11.

0.04 mg each of lipoxygenase (Sigma-Aldrich, derived from Glycine max) and alcohol dehydrogenase (Wako Pure Chemical Industries, Ltd., derived from yeast) were added to the test solution at the same time, and the reaction was allowed to proceed at 5°C for 24 hours. After the reaction was completed, the products in the test solution were analyzed and quantified in the same manner as in Example 1.

As shown in Table 1, the combined yield of isomers such as (E,E isomer) and (E,Z isomer) was 2.2% for 13-oxoODA. The yield of 9-oxoODA was 0.02%.

Comparative Example 2
The same operations and analyses were carried out as in Comparative Example 1, except that the reaction temperature was changed from 5°C to 30°C.

As shown in Table 1, the combined yield of isomers such as (E,E isomer) and (E,Z isomer) was 2.2% for 13-oxoODA. The yield of 9-oxoODA was 0.37%.

Comparative Example 3
As a raw material containing fatty acids, 0.28 g of linoleic acid (manufactured by Wako Pure Chemical Industries, Ltd.) with a purity of 80% was used, to which 0.15 g of potassium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.), 0.05 g of sodium hydrogen carbonate (manufactured by Wako Pure Chemical Industries, Ltd.), and 10 mL of distilled water were added to prepare a test solution. The pH of the solution at this time was 10.2.

0.2 g of soybean powder (variety: Fukuyutaka) and 0.1 g of Mn-containing yeast (5%, Medience Corporation) were added to the test solution and reacted at 5°C for 24 hours. After the reaction was completed, the products in the test solution were analyzed and quantified in the same manner as in Example 1.

As shown in Table 1, the combined yield of isomers such as (E,E isomer) and (E,Z isomer) was 2.9% for 13-oxoODA. The yield of 9-oxoODA was 0.5%.

Comparative Example 4
The same operation and analysis as in Comparative Example 5 were carried out, except that the reaction temperature was changed from 5°C to 30°C.

As shown in Table 1, the combined yield of isomers such as (E,E isomer) and (E,Z isomer) was 1.9% for 13-oxoODA. The yield of 9-oxoODA was 1.1%.

As shown in Table 1, the yields of ketooctadecadienoic acid in Examples 1 to 3 were much higher than the yields of ketooctadecadienoic acid in Comparative Examples 1 to 4. Therefore, it can be seen that the method for producing ketooctadecadienoic acid of the present invention has a significantly excellent effect of producing ketooctadecadienoic acid in high yield.

The soybean powder used in Comparative Examples 3 and 4 is believed to contain both lipoxygenase and alcohol dehydrogenase, and therefore the results of Comparative Examples 3 and 4 are believed to have been obtained under conditions equivalent to the simultaneous addition of lipoxygenase and alcohol dehydrogenase. In Comparative Examples 3 and 4, in addition to this soybean powder, Mn-containing yeast that supplies metals is also added to the test solution. That is, although Comparative Examples 3 and 4 also have the effect of promoting the production reaction of ketooctadecadienoic acid, which is contributed by the metal catalyst, the yields of Comparative Examples 3 and 4 are merely lower than those of Examples 1 to 3. This result also shows that the production method of the present invention has an excellent effect on the conversion yield of ketooctadecadienoic acid from linoleic acid.

Claims (6)

1. A method for producing ketooctadecadienoic acid, comprising: (a) a step of reacting a raw material containing linoleic acid with an enzyme material containing plant-derived lipoxygenase isolated and purified from a plant ; and (b) a step of heating the reaction mixture obtained in step (a) to 70°C or higher and 140°C or lower to inactivate and terminate the enzymatic activity of the lipoxygenase , but not including a step (c) of reacting a product obtained via step (b) with a dehydrogenase. 2. The method for producing ketooctadecadienoic acid according to claim 1 , wherein the raw material containing linoleic acid is a raw material containing fats and oils containing linoleic acid as a constituent component, a raw material containing linoleic acid, or linoleic acid itself. The method for producing ketooctadecadienoic acid according to claim 1 or 2 , wherein the step of reacting the enzyme material is carried out at a temperature of 10°C or higher and 60°C or lower. The method for producing ketooctadecadienoic acid according to any one of claims 1 to 3 , wherein the step of reacting the enzyme material is carried out at a temperature of 20°C or higher and 50°C or lower. 5. The method for producing ketooctadecadienoic acid according to claim 1 , wherein in the step of reacting the enzyme material, the enzyme material is reacted for 0.5 hours or more and 72 hours or less. 2. The method for producing ketooctadecadienoic acid according to claim 1 , wherein the heating is carried out within 0.5 to 24 hours after the enzyme material containing lipoxygenase is allowed to act on the raw material containing linoleic acid.