JP7430995B2 - 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, which are obtained by reacting linoleic acid and oleic acid using enzymes or other means, have attracted attention as so-called rare fatty acids. These rare fatty acids are expected to be used in various industrial applications, especially due to their physiological activity, as interest in people's health and medical care increases. Among them, 13-oxo-octadecadienoic acid and 9-oxo-octadecadienoic acid, which are types of ketooctadecadienoic acid, which are oxo derivatives of linoleic acid, have been found to have activity in improving lifestyle-related diseases such as improving lipid metabolism. As a result, active research is being conducted both domestically and internationally as a functional ingredient that exhibits significant fat-burning effects.

Ketooctadecadienoic acid has long been known to be contained in tomatoes, but its content is extremely small, about 1 ng/mg, and it is used as an active ingredient in foods and medicines. There cannot be a sufficient source of supply for this purpose. Therefore, a source containing a higher content of ketooctadecadienoic acid and a more efficient synthesis of ketooctadecadienoic acid have been desired.

For example, Patent Document 1 describes a method for synthesizing ketooctadecadienoic acid in which lipoxygenase is applied to 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. A method for producing ketooctadecadienoic acid is described, comprising the step of acting an enzyme material comprising:

Japanese Patent Application Publication No. 2016-178913

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 applications, metals may not be present in the product. There is a risk of it being contained. In addition, in order to suppress the production of by-products and improve the yield, in the method for producing ketooctadecadienoic acid described in Patent Document 1, it is essential to set the reaction temperature to less than 20 ° C. In the manufacturing process, complicated equipment and equipment were required for production equipment and temperature control.

Furthermore, in the method for producing ketooctadecadienoic acid described in Patent Document 1, lipoxygenase is used as an enzyme. It has been suggested that dehydrogenase may be added along with lipoxygenase as an enzyme to be used, but since lipoxygenase itself is an enzyme that significantly accelerates the reaction of fatty acids, lipoxygenase produces a large amount of peroxide, especially at reaction temperatures of 20°C or higher. , which further oxidizes the formed ketooctadecanoic acid. Therefore, there is a problem in that the final yield of ketooctadecanoic acid, which is the target substance, decreases. There is a need for a method for producing ketooctadecadienoic acid that safely, stably, and efficiently synthesizes ketooctadecadienoic acid using an enzyme.

The present invention aims to provide a method for producing ketooctadecadienoic acid, which can be produced safely, stably, and efficiently using enzymes, which has hitherto been difficult to supply. purpose.

The present invention includes (a) a step of causing an enzyme material containing lipoxygenase to act on a raw material containing a fatty acid, and (b) a step of stopping the enzymatic reaction of the lipoxygenase contained in the reaction mixture obtained in step (a). , relates to a method for producing ketooctadecadienoic acid.

Furthermore, the present invention relates to a method for producing ketooctadecadienoic acid, which includes the step of (c) allowing a dehydrogenase to act on the product obtained through the step (b).

The raw material containing the fatty acid 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.

A method for producing ketooctadecadienoic acid in which the step of applying the enzyme material is carried out at a temperature of 10°C or higher and 60°C or lower is preferred.

A method for producing ketooctadecadienoic acid in which the step of applying the enzyme material is carried out at a temperature of 20° C. or higher and 50° C. or lower is preferred.

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

A method for producing ketooctadecadienoic acid in which the step of stopping the enzyme reaction is carried out by deactivating the enzymatic activity of the lipoxygenase is preferred.

A method for producing ketooctadecadienoic acid in which the deactivation is performed by heat treatment or pH adjustment is preferred.

A method for producing ketooctadecadienoic acid in which the heat treatment is performed by heating the reaction mixture to a temperature of 70° C. or higher and 140° C. or lower is preferred.

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

A method for producing ketooctadecadienoic acid is preferred, in which the step of stopping the enzymatic reaction is performed by removing the lipoxygenase from the reaction mixture.

A method for producing ketooctadecadienoic acid is preferred, in which the step of stopping the enzyme reaction is carried out by adding an inhibitor of the lipoxygenase to the reaction mixture to inhibit the enzymatic activity of the lipoxygenase.

A method for producing ketooctadecadienoic acid in which the dehydrogenase is an alcohol dehydrogenase is preferred.

A method for producing ketooctadecadienoic acid in which the step of causing the dehydrogenase to act is carried out in the presence of a cofactor is preferred.

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

A method for producing ketooctadecadienoic acid in which the step of causing the dehydrogenase to act is carried out at a temperature of 0° C. or higher and 50° C. or lower is preferred.

A method for producing ketooctadecadienoic acid in which the step of causing the dehydrogenase to act is carried out at a pH of 6 or more and 12 or less is preferred.

A method for producing ketooctadecadienoic acid is preferred, in which the heat treatment is performed within 0.5 to 24 hours after the enzyme material containing lipoxygenase is allowed to act on the raw material containing the fatty acid.

According to the method for producing ketooctadecadienoic acid of the present invention, two different enzymes can be reacted independently and sequentially, so the amount of by-products produced, which has traditionally been a problem in terms of reaction yield, can be reduced. can be significantly suppressed. Therefore, ketooctadecadienoic acid can be efficiently produced. Further, the synthesis temperature can also be set to room temperature, and in particular, ketooctadecadienoic acid can be produced with a 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 in a simpler manner than with conventional techniques.

Ketooctadecadienoic acid is one of the metabolites obtained by oxidative metabolism of linoleic acid in specific microorganisms and plants. Linoleic acid is enzymatically converted into lipid peroxide (hydroperoxyoctadecadienoic acid (HPODE)) by the action of lipoxygenase (LOX), which is one of the enzymes that metabolize linoleic acid. ) is converted to hydroxyoctadecadienoic acid (HODE), which is a hydroxylated fatty acid, non-enzymatically or enzymatically, e.g., by peroxidase, etc., by releasing radicals. Ketooctadecadienoic acid, which is an oxo derivative of a fatty acid, is produced by the action of a dehydrogenase such as alcohol dehydrogenase (ADH) on hydroxyoctadecadienoic acid (HODE).

Specifically, the ketooctadecadienoic acid includes, for example, 9-oxo-10,12-octadecadienoic acid (herein also abbreviated as 9-oxoODA), 13-oxo-9,11 -Octadecadienoic acid (herein also abbreviated as 13-oxoODA), 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 and the like. In addition, when isomers exist in these compounds, all isomers and mixtures thereof are included.

The present invention involves enzymatically converting linoleic acid into hydroperoxyoctadecadienoic acid (HPODE) or hydroxyoctadecadienoic acid (HODE) using lipoxygenase (LOX), then deactivating LOX, and then as needed. Provided is a method for producing ketooctadecadienoic acid with high efficiency by using a dehydrogenase such as ADH. LOX may be deactivated after HPODE is generated or after HODE is generated. EMBODIMENT OF THE INVENTION Hereinafter, the form for implementing this invention is demonstrated in detail. However, the present invention is not limited to the following embodiments.

In some embodiments of the invention, ketooctadecadienoic acid is produced from raw materials that include fatty acids. Examples of raw materials containing fatty acids include raw materials containing oils and fats containing fatty acids as constituent components, raw materials containing fatty acids, fatty acids, and the like. Preferably, the raw material containing a fatty acid is a raw material containing an oil or fat containing linoleic acid as a constituent, 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 esters of linoleic acid.

There are no particular limitations on the raw materials containing fats and oils containing linoleic acid as a constituent, or the raw materials containing linoleic acid. Any raw material containing linoleic acid or linoleic acid can be suitably used in the manufacturing method of this embodiment. If oils and fats containing linoleic acid or plant materials such as soybean, castor, safflower, or corn, or animal materials such as eggs, which are known to have a high content of linoleic acid, are used, the result will be high. It may be preferable to use such raw materials because they can produce ketooctadecadienoic acid in high yields. Further, high purity linoleic acid extracted and/or purified from the above-mentioned raw materials may be used as the raw material.

In some embodiments of the invention, ketooctadecadienoic acid is produced by reacting a raw material containing the fatty acid described above with an enzyme material containing lipoxygenase. As the enzyme material containing lipoxygenase, materials from plants, animals, or bacteria containing lipoxygenase may be used. For example, the lipoxygenase contained in the above-mentioned raw materials is used as it is as the naturally-derived lipoxygenase contained in the enzyme material. You may. From the viewpoint of efficiently proceeding with the oxidation reaction of fatty acids, natural sources, that is, plant-derived, animal-derived materials purified by appropriate isolation and purification techniques from plants such as soybeans, animal bodies such as eggs, or bacteria. or bacterially derived lipoxygenases may be used. Furthermore, lipoxygenase produced as a recombinant by genetic engineering techniques or chemically synthesized lipoxygenase may be used. Furthermore, commercially available products can also be used. Furthermore, the enzyme material may contain other enzymes in addition to lipoxygenase.

The amount of the enzyme material to be used can be appropriately selected depending on the raw material used. For example, an enzyme material is used that contains 100 to 300,000 units of lipoxygenase in terms of purified enzyme per 10 g of linoleic acid contained in the raw material. 2,000 to 180,000 units of lipoxygenase may be used per 10 g of linoleic acid contained in the raw material. Also, 5000 to 90000 units of lipoxygenase may be used. In this specification, 1 unit means that the absorbance of a substrate solution at a wavelength of 234 nm is increased by 0.001 in 1 minute at pH 9 and temperature 25° C. in a 3 mL (width 1 cm) quartz cell using linoleic acid as a substrate. Defined as the amount of enzyme. Therefore, one unit of lipoxygenase activity corresponds to the amount of oxidizing 0.12 μmol of linoleic acid per minute.

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

Note that the reactions or steps described herein can be carried out in a suitable solvent. Appropriate solvents can be readily selected by those skilled in the art. A suitable solvent is one in which the reactions or processes described herein are substantially non-reactive with the reactants, intermediates and/or products at the temperatures as described above at which the reactions are carried out. Additionally, the reactions or steps described herein may be performed in one solvent or a mixture of two or more solvents.

The action time for the enzyme material to act on the fatty acid-containing raw material may be appropriately set depending on the LOX content contained in the enzyme material and the reaction temperature, and is preferably 0.5 to 144 hours, for example. If the action time is shorter than 0.5 hours, the oxidation reaction by LOX may not proceed. When the action time of the enzyme material is longer than 72 hours, side reactions are likely to occur. More preferably, the action time of the enzyme material is about 0.5 to 72 hours. Furthermore, it is desirable that the time be 24 to 72 hours.

The reaction in which the enzyme material containing LOX acts can be carried out within a pH range of approximately 6 to 12. If the pH is less than 6 or more than 12, LOX contained in the enzyme material may be deactivated and/or denatured in the reaction solution. In particular, it is preferable that the reaction in which the enzyme material containing LOX acts is carried out under conditions of pH 9 to 12. Furthermore, it is preferable to deactivate LOX under conditions of pH 10 to 12. If the pH is within such a range, the linoleic acid contained in the raw material may be easily emulsified, and the reaction may be likely to proceed. The pH in the reaction can be adjusted using a known pH adjuster, such as potassium salts such as potassium hydroxide, potassium carbonate, potassium hydrogen carbonate, or potassium hydrogen phosphate, sodium hydroxide, sodium carbonate, and potassium carbonate. This can be carried out using sodium salts such as sodium hydrogen or sodium hydrogen phosphate, amines such as triethylamine, or ammonia.

Oxygen molecules are added to the fatty acids through the reaction of the above-mentioned fatty acid-containing raw material and the lipoxygenase-containing enzyme material to produce peroxidized lipids and/or hydroxylated fatty acids. In the method for producing ketooctadecadienoic acid of the present invention, the enzymatic reaction of lipoxygenase is then stopped in the reaction mixture obtained by the reaction of the fatty acid-containing raw material and the lipoxygenase-containing enzyme material.

The enzymatic reaction of lipoxygenase can be stopped by, for example, deactivating the enzymatic activity of lipoxygenase, although it is not particularly limited. Examples of methods for deactivating the enzymatic activity of lipoxygenase include deactivation of lipoxygenase activity by heat treatment and deactivation of lipoxygenase activity by pH adjustment. Termination of the enzymatic reaction of lipoxygenase can also be achieved by removing the lipoxygenase from the reaction mixture, for example by ultrafiltration or gel filtration, or by adsorbing, binding or encapsulating the enzyme on the surface of a carrier, so-called immobilization. Lipoxygenase is naturally removed from the reaction system by passing it through a reaction tower such as a column using enzyme. Alternatively, it may be carried out by adding to the reaction mixture a reagent such as an inhibitor that interacts with lipoxygenase and selectively inhibits the lipoxygenase reaction. That is, in the present specification, in a broad sense, stopping the enzymatic reaction of lipoxygenase includes not only inactivating the enzymatic activity of lipoxygenase, but also inhibiting the enzymatic activity of lipoxygenase and removing lipoxygenase. shall be provided.

In some embodiments of the invention, termination of the lipoxygenase enzymatic reaction may be performed by inactivating lipoxygenase activity by heat treating the reaction mixture. Deactivation by heat treatment is particularly preferred because it can be easily controlled and efficiently performed even when produced in large quantities. The heating temperature and holding time of the heat treatment can be appropriately set to conditions that can sufficiently deactivate lipoxygenase. For example, the heating temperature is preferably 70°C or higher. If it is preferable to deactivate the enzyme activity of lipoxygenase in a shorter time, it is desirable to set the heating temperature to 90° C. or higher. The upper limit of the heating temperature is not particularly defined, but it is preferably a temperature at which the solvent in the reaction mixture does not volatilize too much, and can be, for example, about the temperature (120 to 140° C.) in general autoclave treatment. By performing the step of stopping the enzymatic reaction of lipoxygenase at this temperature, for example, when the manufactured ketooctadecadienoic acid is used for food or pharmaceutical purposes, the product is heat sterilized at the same time as the lipoxygenase activity is deactivated. Therefore, it may be particularly preferable. The holding time, that is, the lipoxygenase deactivation treatment time is not particularly defined, but generally it can be 5 minutes or more, preferably 10 minutes or more.

When deactivation of lipoxygenase activity is carried out by pH adjustment, the pH of the reaction mixture is adjusted to below pH 6 or above pH 12. The pH can be adjusted using a known pH adjuster such as those described above.

In some embodiments of the invention, the enzymatic reaction of lipoxygenase is stopped by adding a reagent, such as an inhibitor, to the reaction mixture to inhibit the enzymatic activity of lipoxygenase. Lipoxygenase inhibitors include, for example, N-hydroxyurea derivatives, redox inhibitors, and non-redox inhibitors, including, by way of example and not limitation, 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-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), 6-[[3-fluoro-5-(4-methoxyoxy San-4-yl)phenoxy]methyl]-1-methyl-quinolin-2-one (ZD2138) and the like.

In some embodiments of the invention, a dehydrogenase is subsequently applied to the resulting product after the enzymatic reaction of lipoxygenase has been stopped to perform a dehydrogenation reaction. This produces ketooctadecadienoic acid. The dehydrogenase used here is not particularly limited as long as it is an enzyme that can convert the above-mentioned lipid peroxides and hydroxylated fatty acids into oxo derivatives of fatty acids as a substrate. enzyme (ADH). Furthermore, the origin of the enzyme is 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. Furthermore, ADH produced as a recombinant by genetic engineering techniques or chemically synthesized ADH may be used. Furthermore, commercially available ADH can also be used. In order to proceed with the reaction more efficiently, those purified from plants or yeast can be used.

In some embodiments of the invention, it is preferred that the step of acting the dehydrogenase is performed in the presence of a cofactor. Cofactors can be added to the product obtained through a step in which the enzymatic reaction of lipoxygenase is stopped. Cofactors may also be added to the product as a mixture with the enzyme. Cofactors can be selected depending on the type of dehydrogenase. Examples of cofactors can include nicotinamide adenine dinucleotide (NAD ⁺ ), nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), etc. Not limited to these. Preferably, the cofactor is nicotinamide adenine dinucleotide (NAD ⁺ ). The concentration of the cofactor to be added may be such that the dehydrogenation reaction proceeds efficiently.

The amount of ADH to be used can be appropriately selected depending on the raw material used, but for example, 10 to 15,000 units of ADH may be used in terms of purified enzyme per 10 g of linoleic acid contained in the raw material. Also, amounts of ADH greater than 15,000 units may be used. 20-5000 units of ADH may be used, and 100-500 units of ADH may be used. In this specification, 1 unit is defined as the amount of enzyme that converts 1.0 μmol of ethanol into acetaldehyde in 1 minute under conditions of pH 8.8 and 25° C.

The temperature at which ADH acts can be appropriately set depending on the raw material used, but is preferably 0 to 50°C. If the temperature is below 0°C, the solvent may freeze and the reaction may not proceed. Furthermore, at temperatures higher than 50°C, the ADH enzyme is quickly deactivated. More preferably, the temperature may be 10°C or higher and 40°C or lower.

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

The reaction in which ADH acts can preferably be carried out within a pH range of approximately 6 to 12. If the pH is less than 6 or more than 12, ADH may be deactivated 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 the same as in the step of acting with an enzyme material containing LOX, and in this case, it is preferable because there is no need to readjust the pH in the step of acting with ADH. Adjustment of the pH in the reaction can be carried out using a known pH adjuster, for example, potassium salts such as potassium hydroxide, potassium carbonate, potassium hydrogen carbonate or potassium hydrogen phosphate, sodium hydroxide, sodium carbonate, carbonate, etc. This can be carried out using sodium salts such as sodium hydrogen or sodium hydrogen phosphate, amines such as triethylamine, or ammonia.

By acting ADH on the product after the step in which the enzymatic reaction of lipoxygenase has been stopped, lipid peroxide and hydroxylated fatty acid, which are lipoxygenase metabolites of fatty acids produced by the reaction with lipoxygenase, are converted into ketooctadecase. Converted to dienoic 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, it is also possible to deactivate LOX by performing heat treatment after HPODE is generated, and then to obtain ketooctadecadienoic acid by subsequently performing heat treatment. In this case, heat treatment is performed within 0.5 to 24 hours after the enzyme material containing lipoxygenase is applied to the raw material containing fatty acids, and the heat treatment time is desirably 10 minutes or more, preferably 30 minutes or more.

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

As mentioned above, ketooctadecadienoic acid has various isomers such as (E, E form), (Z, E form), (E, Z form), and (Z, Z form). However, their effects as functional components are almost the same, and therefore the yield of ketooctadecadienoic acid described in the following examples is the sum of these isomers.

The method for producing ketooctadecadienoic acid according to the above-described 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 solvent extraction followed by thin layer chromatography or liquid chromatography. Purification methods and conditions may vary depending on the experiment; for example, eluents, solvents, stationary phases, etc. in chromatography and HPLC purification may be selected as appropriate. However, the produced ketooctadecadienoic acid can be used as a reaction solution containing ketooctadecadienoic acid without purification, or as a crude product that has been subjected to a crude purification treatment, such as a product that has been dried after solvent extraction. may be used in

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

In addition, the present inventors have discovered that ketooctadecadienoic acid can be used as a plant activator that exhibits a strong resistance-inducing effect by acting on plants, and has various effects on promoting growth and improving fruit yield. It is known that it has an increasing effect and a disease suppressing effect. For example, specific examples of effective disease control include gray mold on the leaves of cucurbits, such as cucumbers, watermelons, melons, and pumpkins; bacterial wilt, wilt, half-wilt, damping-off, brown root rot, powdery mildew of plants of the Rosaceae family, such as roses and strawberries, scab, gray mold, anthracnose, downy mildew of plants of the Amaranthaceae family, such as spinach, It is effective against black rot, soft rot, bacterial spot disease, Rhizoctonia disease in Brassicaceae such as Chinese cabbage, cabbage, and komatsuna, white silk disease in Apiaceae such as carrots, and blast in Poaceae 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 it is possible to produce ketooctadecadienoic acid, which has been difficult to supply until now, with a sufficient yield, it is expected that it will be useful for analyzing the physiological activity of ketooctadecadienoic acid and for industrial use. Furthermore, the present invention also has the advantage that ketooctadecadienoic acid can be stably produced without the need for the addition of metals, and thus the produced ketooctadecadienoic acid can be used safely in various fields. The method for producing ketooctadecadienoic acid is useful, and the obtained ketooctadecadienoic acid is particularly suitable as a raw material for functional foods, pharmaceuticals, cosmetics, plant activators, and the like.

Further, according to the method for producing ketooctadecadienoic acid according to the embodiment described above, ketooctadecadienoic acid can be efficiently produced at room temperature by independently and sequentially reacting two different enzymes, and special temperature control is required. Since equipment and temperature control are not essential, it is thought that labor-saving, resource-saving, and energy-saving in manufacturing, as well as a reduction in manufacturing costs, can be achieved.

Although the present invention will be explained based on examples, the present invention is not limited to the examples only.

Production 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 to this. In addition, test solutions were prepared. The pH of the test solution at this time was 11.

Add 0.2 mg of lipoxygenase (manufactured by Sigma-Aldrich, derived from Glycine max) to the test solution, react at 30°C for 36 hours, and then place the reaction mixture in a 90°C water bath for 5 minutes to inactivate the enzyme. I let it happen. After the reaction solution was returned to room temperature, 0.2 mg of alcohol dehydrogenase (manufactured by Wako Pure Chemical Industries, Ltd., derived from Yeast) was added, and the reaction solution was further reacted at 30° C. for 24 hours.

The product in the test solution after the completion of the reaction was identified by LC-MS using MS ² spectrum analysis using 13-oxoODA manufactured by Cayman Chemical Co. as a standard substance. Further, quantification was performed using an absolute calibration curve method at a detection wavelength of UV 272 nm. Furthermore, 9-oxoODA was quantified with reference to the absorbance of 13-oxoODA.

As shown in Table 1 as the combined yield of isomers such as (E, E form) and (E, Z form), 13-oxoODA was obtained in a yield of 5.4%. At this time, the yield of 9-oxoODA was 10.9%. Note that the yield (%) was determined based on the following formula.
Yield (%) = (wt% of generated 13-oxoODA or 9-oxoODA)/(initial wt% of raw material linoleic acid used)

・Example 2
The operation and analysis were performed in the same manner as in Example 1, except that the reaction time of the test solution with lipoxygenase was changed to 24 hours instead of 36 hours.

As shown in Table 1, 13-oxoODA was obtained with a yield of 3.5% as a combined yield of isomers such as (E, E form) and (E, Z form). At this time, 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.) and dipotassium hydrogen phosphate ( A test solution was prepared by adding 23.3 g (manufactured by Wako Pure Chemical Industries, Ltd.) and 300 mL of distilled water. The pH of the test solution at this time was 9.0.

Add 0.4 mg of lipoxygenase (manufactured by Sigma-Aldrich, derived from Glycine max) to the test solution, react at 15°C for 3 hours, and then place the reaction mixture in a 90°C water bath for 90 minutes to inactivate the enzyme. I let it happen. The product after the completion of the reaction was operated and analyzed in the same manner as in Example 1.

As shown in Table 1, 13-oxoODA was obtained with a yield of 2.6% as a combined yield of isomers such as (E, E form) and (E, Z form). At this time, the yield of 9-oxoODA was 1.2%.

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

Lipoxygenase (manufactured by Sigma-Aldrich, derived from Glycine max) and alcohol dehydrogenase (manufactured by Wako Pure Chemical Industries, Ltd., derived from Yeast) were added at the same time to the test solution in an amount of 0.04 mg each, and the mixture was allowed to react at 5°C for 24 hours. . The product in the test solution after the completion of the reaction was analyzed and quantified in the same manner as in Example 1.

As shown in Table 1, 13-oxoODA was obtained with a yield of 2.2% as a combined yield of isomers such as (E, E form) and (E, Z form). At this time, the yield of 9-oxoODA was 0.02%.

・Comparative example 2
The operation and analysis were performed in the same manner as in Comparative Example 1 except that the reaction temperature was changed to 30°C instead of 5°C.

As shown in Table 1, 13-oxoODA was obtained with a yield of 2.2% as a combined yield of isomers such as (E, E form) and (E, Z form). At this time, 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, along with 0.15 g of potassium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) and sodium hydrogen carbonate (manufactured by Wako Pure Chemical Industries, Ltd.). A test solution was prepared by adding 0.05 g (manufactured by Yaku Kogyo Co., Ltd.) and 10 mL of distilled water. 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%, manufactured by Medience) were added to the test solution, and the mixture was reacted at 5° C. for 24 hours. The product in the test solution after the completion of the reaction was analyzed and quantified in the same manner as in Example 1.

As shown in Table 1, 13-oxoODA was obtained with a yield of 2.9% as a combined yield of isomers such as (E, E form) and (E, Z form). At this time, the yield of 9-oxoODA was 0.5%.

・Comparative example 4
The operation and analysis were performed in the same manner as in Comparative Example 5, except that the reaction temperature was changed to 30°C instead of 5°C.

As shown in Table 1, 13-oxoODA was obtained with a yield of 1.9% as a combined yield of isomers such as (E, E form) and (E, Z form). At this time, 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 those in Comparative Examples 1 to 4. Therefore, it can be seen that the method for producing ketooctadecadienoic acid of the present invention has a remarkable effect of producing ketooctadecadienoic acid in high yield.

In addition, since the soybean powder used in Comparative Examples 3 and 4 is thought to contain both lipoxygenase and alcohol dehydrogenase, the results of Comparative Examples 3 and 4 are based on the content of lipoxygenase and alcohol dehydrogenase. It is thought that this was obtained under the same conditions as simultaneous addition. In Comparative Examples 3 and 4, in addition to this soybean powder, Mn-containing yeast that supplies metals was also added to the test solution. That is, in Comparative Examples 3 and 4, although the metal catalyst contributed to the effect of promoting the production reaction of ketooctadecadienoic acid, the yields of Comparative Examples 3 and 4 were lower than those of Examples 1 to 3. It's just low in comparison. 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 (17)

(a) a step of causing an enzyme material containing plant-derived lipoxygenase to act on a raw material containing linoleic acid ;
(b) stopping the enzymatic reaction of the lipoxygenase contained in the reaction mixture obtained in step (a) ; and
(c) A step of causing a dehydrogenase to act on the product obtained through the step (b) above.
A method for producing ketooctadecadienoic acid, comprising: The method for producing ketooctadecadienoic acid according to claim 1 , wherein the raw material containing linoleic acid is a raw material containing an oil or fat containing linoleic acid as a constituent, a raw material containing linoleic acid, or linoleic acid. The method for producing ketooctadecadienoic acid according to claim 1 or 2 , wherein the step of causing the enzyme material to act 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 causing the enzyme material to act is carried out at a temperature of 20°C or higher and 50°C or lower. The method for producing ketooctadecadienoic acid according to any one of claims 1 to 4 , wherein in the step of allowing the enzyme material to act, the enzyme material is allowed to act for 0.5 hours or more and 72 hours or less. The method for producing ketooctadecadienoic acid according to any one of claims 1 to 5 , wherein the step of stopping the enzyme reaction is carried out by deactivating the enzyme activity of the lipoxygenase. The method for producing ketooctadecadienoic acid according to claim 6 , wherein the deactivation is performed by heat treatment or pH adjustment. 8. The method for producing ketodecadienoic acid according to claim 7 , wherein the heat treatment is performed by heating the reaction mixture to a temperature of 70°C or higher and 140°C or lower. 8. The method for producing ketooctadecadienoic acid according to claim 7 , wherein the pH adjustment is performed by adjusting the reaction mixture to a pH of less than 6 or more than 12. The method for producing ketooctadecadienoic acid according to any one of claims 1 to 5 , wherein the step of stopping the enzyme reaction is performed by removing the lipoxygenase from the reaction mixture. The ketooctadecadiene according to any one of claims 1 to 5 , wherein the step of stopping the enzyme reaction is carried out by adding an inhibitor of the lipoxygenase to the reaction mixture to inhibit the enzymatic activity of the lipoxygenase. Acid production method. The method for producing ketooctadecadienoic acid according to any one of claims 1 to 11 , wherein the dehydrogenase is an alcohol dehydrogenase. The method for producing ketooctadecadienoic acid according to any one of claims 1 to 12 , wherein the step of causing the dehydrogenase to act is carried out in the presence of a cofactor. The method for producing ketooctadecadienoic acid according to claim 13 , wherein the cofactor is NAD ⁺ . The method for producing ketooctadecadienoic acid according to any one of claims 1 to 14 , wherein the step of allowing the dehydrogenase to act is carried out at a temperature of 0°C or higher and 50°C or lower. The method for producing ketooctadecadienoic acid according to any one of claims 1 to 15 , wherein the step of causing the dehydrogenase to act is carried out at a pH of 6 or more and 12 or less. The method for producing ketooctadecadienoic acid according to claim 7 , wherein the heat treatment is performed within 0.5 to 24 hours after the enzyme material containing lipoxygenase is applied to the raw material containing linoleic acid.