Process for manufacture of compositions containing processed unsaturated fatty acids and their use

Abstract

The present invention addresses the problem of providing a method for producing unsaturated fatty acids having different carbon chain lengths while maintaining the double bond moiety of the unsaturated fatty acid. The present invention relates to a method for producing a composition containing a processed unsaturated fatty acid, comprising a step of causing a fatty acid alpha-position hydrooxidase to act on a composition containing an unsaturated long-chain fatty acid or a composition containing a compound having an unsaturated long-chain fatty acid in a partial structure. The present invention also relates to a method for shortening the chain length of an unsaturated long-chain fatty acid, a method for producing a processed unsaturated fatty acid-containing food, and an enzyme agent for shortening the chain length of an unsaturated long-chain fatty acid.

Description

Technical Field

[0001] The present invention relates to a method for producing a composition containing processed unsaturated fatty acids and their uses. Specifically, the present invention relates to a method for producing a composition containing processed unsaturated fatty acids, a method for shortening the chain length of unsaturated long-chain fatty acids, a method for producing a food containing processed unsaturated fatty acids, and an enzyme for shortening the chain length of unsaturated long-chain fatty acids. Background Art

[0002] The chemical synthesis of fatty acids is an extremely difficult technology, and most commercially available fatty acids are extracted from oil and fat raw materials. Synthesis of fatty acids with different unsaturation positions is nearly impossible, and only a limited number of biosynthetically produced unsaturated fatty acids, such as oleic acid, palmitoleic acid, myristoleic acid, and linoleic acid, are extracted and sold.

[0003] Furthermore, the majority of fatty acids in mammals and birds have even numbers of carbon atoms, with only a small number of odd-numbered fatty acids occurring naturally. The functions and commercial value of odd-numbered fatty acids have not been fully understood, and there are few methods for their artificial synthesis.

[0004] In recent years, in order to shorten the chain length of saturated fatty acids, various technologies have been studied. For example, a fatty acid α-position hydroxylase (cytochrome P450) derived from Sphingomonas paucimobilis is disclosed in non-patent literature 1, and the chain length of saturated fatty acids is shortened by hydroxylating the α-position of fatty acids. A method for obtaining saturated fatty acids with shorter chain lengths such as nonanoic acid by acting on myristic acid (saturated fatty acids) with cytochrome P450 derived from Exiguobacterium sp. is disclosed in non-patent literature 2. In addition, a method for manufacturing a triglyceride containing odd-number fatty acids as a main component and a triglyceride containing highly unsaturated fatty acids as a main component is disclosed in patent literature 1.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Laid-Open No. 2016-89025

[0008] Non-patent literature

[0009] Non-patent document 1: FEBS Letters 386 (1996) 252-254

[0010] Non-patent literature 2: Catalysis Science & Technology, 2018, 8, 434-442 Summary of the Invention

[0011] As mentioned above, methods for shortening the chain length of saturated fatty acids have been studied. However, methods for shortening the chain length of unsaturated fatty acids have not been studied. Unsaturated fatty acids have various effects, such as lowering blood cholesterol, and it is necessary to establish a technology for changing the chain length of unsaturated fatty acids.

[0012] Therefore, an object of the present invention is to provide a method for producing unsaturated fatty acids having different carbon chain lengths while maintaining the double bond portion of the unsaturated long-chain fatty acid.

[0013] Specific examples of the present invention are shown below.

[0014] [1] A method for producing a composition containing processed unsaturated fatty acids, comprising the steps of allowing a fatty acid α-hydroxylase to act on a composition containing unsaturated long-chain fatty acids or a composition containing a compound having an unsaturated long-chain fatty acid as a partial structure.

[0015] [2] The method for producing a processed unsaturated fatty acid-containing composition according to [1], wherein the fatty acid α-hydroxysuccinyltransferase is cytochrome P450.

[0016] [3] The method for producing a processed unsaturated fatty acid-containing composition according to [1] or [2], wherein the fatty acid α-hydroxysuccinyltransferase is composed of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence equivalent thereto.

[0017] [4] The method for producing a processed unsaturated fatty acid-containing composition according to any one of [1] to [3], wherein the unsaturated long-chain fatty acid-containing composition contains at least one selected from the group consisting of myristoleic acid, palmitoleic acid, and oleic acid.

[0018] [5] The method for producing a processed unsaturated fatty acid-containing composition according to any one of [1] to [4], wherein the composition containing a compound having an unsaturated long-chain fatty acid as a partial structure is a glyceride-containing composition.

[0019] [6] A method for shortening the chain length of an unsaturated long-chain fatty acid, comprising the following steps: allowing a fatty acid α-hydroxylase to act on an unsaturated long-chain fatty acid or a compound having an unsaturated long-chain fatty acid in a partial structure.

[0020] [7] A method for producing a food containing processed unsaturated fatty acids, comprising the steps of allowing fatty acid α-hydroxylase to act on a food containing unsaturated long-chain fatty acids or a food containing a compound having unsaturated long-chain fatty acids in a partial structure.

[0021] [8] An enzyme for shortening the chain length of unsaturated long-chain fatty acids, comprising fatty acid α-hydroxylase.

[0022] [9] The enzyme agent for shortening the chain length of unsaturated long-chain fatty acids according to [8], wherein the fatty acid α-hydroxylase is cytochrome P450.

[0023]

[10] The enzyme for shortening the chain length of unsaturated long-chain fatty acids according to [8] or [9], wherein the fatty acid α-hydroxylase is composed of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence equivalent thereto.

[0024]

[11] The enzyme agent for shortening the chain length of unsaturated long-chain fatty acids according to any one of [8] to

[10] , wherein the unsaturated long-chain fatty acid is at least one selected from myristoleic acid, palmitoleic acid, and oleic acid.

[0025] [A] A processed unsaturated fatty acid-containing composition produced by the method for producing a processed unsaturated fatty acid-containing composition according to any one of [1] to [5].

[0026] [B] The processed unsaturated fatty acid-containing composition according to [A], further comprising an odd-numbered unsaturated fatty acid.

[0027] According to the present invention, a processed unsaturated fatty acid-containing composition can be obtained by shortening the chain length of unsaturated long-chain fatty acids. The processed unsaturated fatty acid-containing composition contains unsaturated fatty acids having different carbon chain lengths while retaining the double bond portion of the unsaturated long-chain fatty acids. Furthermore, the present invention provides a method for shortening the chain length of unsaturated long-chain fatty acids and an enzyme for shortening the chain length of unsaturated long-chain fatty acids. DETAILED DESCRIPTION

[0028] Hereinafter, the present invention will be described in detail. Although the description of the constituent elements described below may be based on representative embodiments and specific examples, the present invention is not limited to such embodiments.

[0029] In this specification, the 20 kinds of amino acid residues in the amino acid sequence are sometimes represented by single-letter abbreviations. In this case, glycine (Gly) is G, alanine (Ala) is A, valine (Val) is V, leucine (Leu) is L, isoleucine (Ile) is I, phenylalanine (Phe) is F, tyrosine (Tyr) is Y, tryptophan (Trp) is W, serine (Ser) is S, threonine (Thr) is T, cysteine ​​(Cys) is C, methionine (Met) is M, aspartic acid (Asp) is D, glutamic acid (Glu) is E, asparagine (Asn) is N, glutamine (Gln) is Q, lysine (Lys) is K, arginine (Arg) is R, histidine (His) is H, and proline (Pro) is P. In addition, in this specification, the left end of the amino acid sequence shown is N-terminal, and the right end is C-terminal.

[0030] As used herein, "non-polar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. "Non-charged amino acids" include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. "Acidic amino acids" include aspartic acid and glutamic acid. "Basic amino acids" include lysine, arginine, and histidine.

[0031] In this specification, "substitution" includes not only the case where an amino acid residue is artificially introduced but also the case where an amino acid residue is naturally introduced, that is, the amino acid residue is originally different. In this specification, amino acid residue substitution can be artificial substitution or natural substitution, preferably artificial substitution.

[0032] (Method for producing a composition containing processed unsaturated fatty acids)

[0033] The present embodiment relates to a method for producing a processed unsaturated fatty acid composition, which comprises the following steps: allowing a fatty acid α-hydroxylase to act on a composition containing an unsaturated long-chain fatty acid composition or a composition containing a compound having an unsaturated long-chain fatty acid in a partial structure (hereinafter, in this specification, the composition containing an unsaturated long-chain fatty acid composition and the composition containing a compound having an unsaturated long-chain fatty acid in a partial structure are collectively referred to as "the composition containing unsaturated long-chain fatty acids"). In the present embodiment, by allowing a fatty acid α-hydroxylase to act on a composition containing an unsaturated long-chain fatty acid, an unsaturated long-chain fatty acid with a longer chain length can be shortened to obtain a fatty acid with a shorter carbon chain length than the carbon chain length of the original unsaturated long-chain fatty acid. In addition, in the present embodiment, a fatty acid with a shorter carbon chain length can be obtained while the unsaturated bond portion of the unsaturated long-chain fatty acid remains unchanged.

[0034] In the past, it was thought that unsaturated fatty acids were owing to having rigid double bonds in structure, and therefore compared with saturated fatty acids, were difficult to make their short-chain because of being hindered by contact with enzyme. However, the inventor has found that unsaturated fatty acids have a higher reactivity than saturated fatty acids. And the inventor has found that can generate natural odd number unsaturated fatty acids (C17:1, C15:1, C13:1, C11:1, C9:1, C7:1, C5:1, C3:1) that hardly do not exist, synthesize difficulty. That is, in the manufacture method of present embodiment, can obtain containing the processing unsaturated fatty acid composition that contains odd number fatty acids, and then can obtain containing the processing unsaturated fatty acid composition that contains odd number unsaturated fatty acids.

[0035] It should be noted that, in this specification, when an unsaturated fatty acid is represented as Cx:y, it means an unsaturated fatty acid having x carbon atoms and y carbon-carbon double bonds. For example, C17:1 represents an unsaturated fatty acid having 17 carbon atoms and 1 carbon-carbon double bond.

[0036] In addition, in the present embodiment, unsaturated fatty acids with different double bond moieties can be obtained. Here, "unsaturated fatty acids with different double bond moieties" refer to unsaturated fatty acids having the same number of carbon atoms as exemplified by the following structural formula and having different lengths (number of carbon atoms) from the carboxyl end to the double bond moiety. In the present embodiment, since fatty acid α-hydroxylase hydroxylates the α-position of unsaturated long-chain fatty acids and shortens the fatty acid chain length, the unsaturated fatty acids generated by shortening become substances with different double bonds even if the degree of unsaturation is the same. Since unsaturated fatty acids with different double bond moieties do not exist in nature, a technology that can obtain such unsaturated fatty acids is useful.

[0037]

[0038] <Unsaturated long-chain fatty acid-containing composition>

[0039] The composition containing unsaturated long-chain fatty acids contains unsaturated long-chain fatty acids. Furthermore, the composition containing a compound partially containing an unsaturated long-chain fatty acid contains a compound partially containing an unsaturated long-chain fatty acid. Fatty acids can be categorized as unsaturated and saturated fatty acids. Unsaturated fatty acids are fatty acids with one or more carbon-carbon double bonds. Monounsaturated fatty acids refer to unsaturated fatty acids with only one carbon-carbon double bond. On the other hand, polyunsaturated fatty acids refer to unsaturated fatty acids with two or more carbon-carbon double bonds and are categorized as ω6 and ω3 types. Specific examples include monounsaturated fatty acids such as myristic acid, palmitoleic acid, oleic acid, gondoic acid, erucic acid, nervonic acid, hexacosenoic acid, and octacosenoic acid; ω3 polyunsaturated fatty acids such as α-linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, and docosahexaenoic acid; and ω6 polyunsaturated fatty acids such as linoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, and docosapentaenoic acid. Fatty acids are classified into long-chain, medium-chain, and short-chain fatty acids based on their chain length. Long-chain fatty acids are fatty acids with 11 or more carbon atoms, medium-chain fatty acids are fatty acids with 7 to 10 carbon atoms, and short-chain fatty acids are fatty acids with 6 or fewer carbon atoms.

[0040] Examples of the unsaturated long-chain fatty acids contained in the unsaturated long-chain fatty acid-containing composition include unsaturated fatty acids having 11 or more carbon atoms. The unsaturated fatty acids contained in the unsaturated fatty acid-containing composition are not particularly limited as long as the target fatty acid can be obtained, and may be polyunsaturated fatty acids or monounsaturated fatty acids. Examples of unsaturated long-chain fatty acids include myristic acid, palmitoleic acid, oleic acid, gondoic acid, erucic acid, nervonic acid, hexacosenoic acid, octacosenoic acid, linoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, docosapentaenoic acid, α-linolenic acid, octadecatetraenoic acid, eicosatetraenoic acid, eicosapentaenoic acid, and docosahexaenoic acid. As unsaturated long-chain fatty acids, unsaturated long-chain odd-numbered fatty acids other than the above-mentioned fatty acids may also be used.

[0041] Wherein, unsaturated long-chain fatty acid is preferably monounsaturated fatty acid, more preferably contains at least one selected from myristoleic acid, palmitoleic acid, oleic acid, gondoic acid, erucic acid, nervonic acid, hexacosenoic acid and octacosenoic acid, further preferably contains at least one selected from myristoleic acid, palmitoleic acid and oleic acid. In addition, in the present embodiment, even when using unsaturated long-chain fatty acids with more than 16 carbon atoms, short-chaining of fatty acids can be carried out. That is, in the present embodiment, unsaturated long-chain fatty acids are also preferably selected from at least one selected from palmitoleic acid, oleic acid, gondoic acid, erucic acid, nervonic acid, hexacosenoic acid and octacosenoic acid, further preferably at least one selected from palmitoleic acid and oleic acid. It should be noted that the unsaturated long-chain fatty acids contained in the unsaturated long-chain fatty acid-containing composition can be one alone or can include multiple types.

[0042] In this embodiment, when a composition containing a compound having an unsaturated long-chain fatty acid as a substrate is used, the compound having an unsaturated long-chain fatty acid as a substrate is preferably a fatty acid ester, more preferably a glyceride. That is, the composition containing a compound having an unsaturated long-chain fatty acid as a substrate is preferably a fatty acid ester composition, more preferably a glyceride composition. As an example of a glyceride composition, oils and fats can be mentioned. The glyceride composition contains glycerides, and as glycerides, triglycerides, diglycerides, and monoglycerides can be used. It should be noted that glycerides are formed by the combination of at least one unsaturated long-chain fatty acid and glycerol.

[0043] In the present embodiment, containing the unsaturated long-chain fatty acid composition, in addition to the unsaturated long-chain fatty acids, also can contain other arbitrary components, also can be made of unsaturated long-chain fatty acids. As other arbitrary components, for example, saturated fatty acids, glycerol, salts (buffer salts), hydrogen peroxide etc. can be enumerated. In addition, containing the unsaturated fatty acid composition can also be the food or food material that contains the compound of unsaturated long-chain fatty acids, partial structure with unsaturated long-chain fatty acids. As such food or food material, edible oil (olive oil, safflower oil, sunflower oil, rapeseed oil, peanut oil and rice bran oil etc.), meat (beef, chicken and pork etc.), cheese, nuts (macadamia nuts, almonds etc.), peanuts, sunflower seeds, eggs, milk, olives and avocado etc. can be enumerated.

[0044] In addition, the unsaturated long-chain fatty acid-containing composition may also contain a pH adjuster, a preservative, a stabilizer, a thickener, an antioxidant, a surfactant, an emulsifier, a solubilizer, a solubilizing agent, a bulking agent, a suspending agent, an unsaturated fatty acid (excluding the above-mentioned unsaturated long-chain fatty acids), a saturated fatty acid, a salt (buffer salt), hydrogen peroxide, etc. as other optional components. Examples of the pH adjuster include acetate buffer, phosphate buffer, MES buffer, HEPES buffer, PIPES buffer, Bis-tris buffer, MOPS buffer, etc.

[0045] The content of the unsaturated long-chain fatty acids contained in the unsaturated long-chain fatty acid composition is preferably 0.001 mass % or more, more preferably 0.002 mass % or more, further preferably 0.01 mass % or more, more preferably 0.1 mass % or more, further preferably 1 mass % or more, and then more preferably 5 mass % or more, particularly preferably 10 mass % or more. In addition, the content of the unsaturated long-chain fatty acids can be 100 mass % or less, 90 mass % or less, 80 mass % or less, 70 mass % or less, 60 mass % or less, or 50 mass % or less relative to the gross mass of the unsaturated long-chain fatty acid composition.

[0046] The form of the unsaturated long-chain fatty acid-containing composition is not particularly limited as long as the enzyme can react, and may be in any form of powder, solid, gel, or liquid (paste). Among them, the unsaturated long-chain fatty acid-containing composition is preferably in liquid (paste).

[0047] <Fatty acid α-hydroxylase>

[0048] In this embodiment, a fatty acid α-hydroxylase is allowed to act on a composition containing unsaturated long-chain fatty acids. The fatty acid α-hydroxylase is not particularly limited in type or source, as long as it is an enzyme that oxidizes the α-position of fatty acids. Examples of fatty acid α-hydroxylase include oxygenase and peroxidase.

[0049] From the perspective of obtaining a shortening effect on fatty acids, the fatty acid α-hydroxylase is preferably an oxygenase, and more preferably a cytochrome P450. The type of cytochrome P450 is not particularly limited, and examples thereof include P450BSβ (CYP152A1) derived from Bacillus subtilis, P450Exα (CYP152N1) derived from Exiguobacterium sp., P450CLA (CYP152A2) derived from Clostridium acetobutylicum, and P450SPα (CYP152B1) derived from Sphingomonas paucimobilis. In this embodiment, the fatty acid α-hydroxylase is preferably a fatty acid α-hydroxylase in which the α-hydroxyl action is dominant over the β-oxidation action. Examples of such fatty acid α-hydroxylase include P450Exα (CYP152N1) derived from Exiguobacterium and P450SPα (CYP152B1) derived from Sphingomonas paucimobilis. Cytochrome P450 (P450Exα) derived from Exiguobacterium is more preferred. These fatty acid α-hydroxylase enzymes may be used alone or in combination.

[0050] The fatty acid α-hydroxylation enzyme is preferably an enzyme consisting of the amino acid sequence of sequence number 1 or an amino acid sequence equivalent thereto. Here, "equivalent amino acid sequence" refers to an amino acid sequence that differs from the amino acid sequence of the benchmark (the amino acid sequence of sequence number 1) in a part but the difference does not substantially affect the function of the protein (here, the fatty acid α-hydroxylation ability). Therefore, the enzyme with an equivalent amino acid sequence catalyzes the fatty acid α-hydroxylation reaction. The degree of activity is not particularly limited as long as it can function as a fatty acid α-hydroxylation enzyme. Among them, the activity is preferably the same as or higher than that of the enzyme consisting of the amino acid sequence as the benchmark. In addition, "differences in a part of the amino acid sequence" are produced, for example, by the deletion, substitution, addition, insertion of one or more amino acids in the amino acids constituting the amino acid sequence, or any combination thereof. Differences in a part of the amino acid sequence are acceptable as long as the fatty acid α-hydroxylation activity is maintained (it may be a slight change in activity). As long as this condition is met, the position where the difference in the amino acid sequence exists is not particularly limited. In addition, the difference in the amino acid sequence can occur at multiple sites (positions).

[0051] The number of amino acids that contribute to the partial difference in the amino acid sequence is preferably equivalent to, for example, less than about 30% of all amino acids constituting the amino acid sequence, more preferably less than about 20%, even more preferably less than about 15%, even more preferably less than about 10%, even more preferably less than about 7%, even more preferably less than about 5%, particularly preferably less than about 3%, even more preferably less than about 2%, and most preferably less than about 1%. Thus, an enzyme composed of an equivalent amino acid sequence has, for example, about 70% or more, preferably about 80% or more, more preferably about 85% or more, about 90% or more, about 93% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more homology to the reference amino acid sequence.

[0052] One typical example of "difference in a part of the amino acid sequence" is the mutation (change) in the amino acid sequence by deletion, substitution, addition or insertion of 1 to 40 (preferably 1 to 30, more preferably 1 to 10, further preferably 1 to 7, even more preferably 1 to 5, even more preferably 1 to 3) amino acids among the amino acids constituting the amino acid sequence, or a combination thereof.

[0053] For example, an equivalent amino acid sequence can be obtained by conservative amino acid substitution in an amino acid residue that is not essential for the α-hydroxylation ability of fatty acids. "Conservative amino acid substitution" here refers to replacing a certain amino acid residue with an amino acid residue having a side chain of the same nature. Amino acid residues are classified into several families according to their side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions are preferably substitutions between amino acid residues within the same family.

[0054] The homology (%) between two amino acid sequences can be determined, for example, by the following steps. First, the two sequences are aligned in a manner that allows for optimal comparison (for example, gaps can be introduced into the first sequence to optimize alignment with the second sequence). When the molecule (amino acid residue) at a particular position in the first sequence is identical to the molecule at the corresponding position in the second sequence, the molecules at that position are said to be identical. The homology between two sequences is a function of the number of identical positions shared by the two sequences (i.e., homology (%) = number of identical positions / total number of positions × 100), preferably also taking into account the number and size of the gaps required for optimal alignment.

[0055] The comparison of two sequences and the determination of homology can be achieved using a mathematical algorithm. As a specific example of a mathematical algorithm that can be used for sequence comparison, there is an algorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-68 and improved in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-77, but it is not limited thereto. The homology of amino acid sequences can be obtained, for example, by blastp (protein-protein BLAST) of the National Center for Biotechnology Information (NCBI). The parameters can be used with the default parameters, for example, by using the BLOSUM62 matrix and setting Gap Costs to Existence: 11 and Extension: 1.

[0056] The fatty acid α-hydroxylase may be part of a larger protein (e.g., a fusion protein). Examples of sequences added to the fusion protein include sequences that facilitate purification, such as multiple histidine residues, and sequences that ensure stability during recombinant production.

[0057] The amount of fatty acid α-hydroxylase used in the step of allowing the fatty acid α-hydroxylase to act on the unsaturated long-chain fatty acid-containing composition is not particularly limited, but is preferably, for example, 0.01 U or more, more preferably 0.05 U or more, 0.1 U or more, 0.5 U or more, 1 U or more, 5 U or more, 10 U or more, 50 U or more, 100 U or more, 500 U or more, 1000 U or more, 5000 U or more, 10,000 U or more, or 30,000 U or more per mg of the unsaturated long-chain fatty acid. The amount of fatty acid α-hydroxylase used per mg of the unsaturated long-chain fatty acid is, for example, preferably 1,000,000 U or less, more preferably 100,000 U or less, 60,000 U or less, 40,000 U or less, 20,000 U or less, 10,000 U or less, 5,000 U or less, 2,000 U or less, 1,000 U or less, or 100 U or less.

[0058] The activity of fatty acid α-hydroxylase can be determined by measuring the change in absorbance at 469 nm when treated with a 5 mM 2,6-dimethoxyphenol solution (0.1 M potassium phosphate, 0.5 M potassium acetate buffer (pH 7.0), 0.5 mM hydrogen peroxide) at 40°C as a substrate. In this specification, 1 unit (1 U) is defined as the amount of enzyme that oxidizes 1 nmol of substrate per minute.

[0059] <Optional ingredients>

[0060] In the present embodiment, in order to further improve the effect of the present invention, in addition to the above-mentioned fatty acid α-position hydroxylase, hydrogen peroxide can also be used. In this case, hydrogen peroxide itself can be added during the reaction, and an enzyme (oxidase, etc.) that generates hydrogen peroxide can also be used. In addition, hydrogen peroxide can also be added in advance to the unsaturated long-chain fatty acid-containing composition.

[0061] The amount of hydrogen peroxide used is not particularly limited. For example, the amount of hydrogen peroxide added per 1 mg of unsaturated long-chain fatty acid is preferably 0.01 mg or more, 0.1 mg or more, 0.5 mg or more, 1 mg or more, or 5 mg or more. From the perspective of further improving the effect, it is more preferably 7 mg or more or 10 mg or more. For example, the amount of hydrogen peroxide added per 1 mg of unsaturated long-chain fatty acid is preferably 10 g or less, 1 g or less, 500 mg or less, 200 mg or less, 100 mg or less, or 50 mg or less.

[0062] The amount of hydrogen peroxide added per 1 U of fatty acid α-hydroxylase is preferably, for example, 0.001 μg or more, 0.01 μg or more, 0.05 μg or more, 0.1 μg or more, 0.2 μg or more, or 0.3 μg or more, and more preferably 1.5 μg or more from the viewpoint of further improving the effect. The amount of hydrogen peroxide added per 1 U of fatty acid α-hydroxylase is preferably, for example, 10 mg or less, 1 mg or less, 0.5 mg or less, 0.2 mg or less, 0.1 mg or less, 0.05 mg or less, 0.01 mg or less, or 0.005 mg or less.

[0063] In the present embodiment, oxidase can be used instead of hydrogen peroxide. As oxidase, as long as it is an enzyme that generates hydrogen peroxide by reacting with an oxidized substrate, its type and source are not particularly limited. As examples of oxidase, sugar oxidase with a wide range of sugars including glucose as substrate, glucose oxidase with glucose as substrate, sugar alcohol oxidase with glycerol as substrate, oxalate oxidase with oxalic acid as substrate can be cited. Among them, oxidase is preferably sugar oxidase or glucose oxidase, more preferably sugar oxidase. The type of sugar oxidase is not particularly limited, for example, sugar oxidase from Acremonium chrysogenum can be cited. When using oxidase, substrate can be added as needed.

[0064] The amount of oxidase used is not particularly limited. When using carbohydrate oxidase, the amount of carbohydrate oxidase added per mg of unsaturated long-chain fatty acids is preferably, for example, 0.01 U or more, 0.05 U or more, 0.1 U or more, 0.5 U or more, 1 U or more, 2 U or more, or 4 U or more. Furthermore, the amount of carbohydrate oxidase added per mg of unsaturated long-chain fatty acids is preferably, for example, 5000 U or less, 1000 U or less, 500 U or less, 200 U or less, 100 U or less, 50 U or less, 40 U or less, 30 U or less, or 20 U or less.

[0065] When using an oxidase, the amount of the oxidase substrate added is not particularly limited. The amount of the oxidase substrate added per mol of the unsaturated long-chain fatty acid is preferably, for example, 0.1 mol or more, 0.5 mol or more, 1 mol or more, 2 mol or more, 5 mol or more, 7 mol or more, or 10 mol or more. Furthermore, the amount of the oxidase substrate added per mol of the unsaturated long-chain fatty acid is preferably, for example, 1000 mol or less, 500 mol or less, 100 mol or less, 50 mol or less, or 30 mol or less.

[0066] When glucose is used as a substrate for carbohydrate oxidase, the amount of glucose added per mol of fatty acid is preferably, for example, 0.1 mol or more, 0.5 mol or more, 1 mol or more, 5 mol or more, 10 mol or more, 15 mol or more, or 20 mol or more. Furthermore, the amount of glucose added per mol of fatty acid is preferably, for example, 1000 mol or less, 500 mol or less, 100 mol or less, 70 mol or less, 50 mol or less, or 30 mol or less.

[0067] The amount of glucose added per 1 mg of unsaturated long-chain fatty acids is preferably, for example, 0.1 mg or more, 0.5 mg or more, 1 mg or more, 2 mg or more, 5 mg or more, 7 mg or more, or 10 mg or more. The amount of glucose added per 1 mg of unsaturated long-chain fatty acids is preferably, for example, 1000 mg or less, 500 mg or less, 100 mg or less, 50 mg or less, 30 mg or less, or 20 mg or less.

[0068] When using carbohydrate oxidase or glucose oxidase as the oxidase, the activity of the carbohydrate oxidase or glucose oxidase can be measured using the following method. First, an appropriate amount of enzyme is measured and dissolved or uniformly dispersed in cooled potassium phosphate / sodium hydroxide buffer (0.1 mol / L) at pH 7.0 to 50 mL. The resulting solution is used as the sample solution. 2.50 g of D(+)-glucose is measured and dissolved in water to 25 mL. The resulting solution is used as the substrate solution. 0.5 mL of the substrate solution, 2 mL of potassium phosphate / sodium hydroxide buffer (0.1 mol / L, pH 7.0, containing phenol), 0.5 mL of a peroxidase test solution (25 units / mL), and 0.1 mL of a 4-aminoantipyrine solution (0.4 mass %) are placed in a quartz cuvette and heated at 37°C for 10 minutes. Separately, 0.1 mL of the sample solution is added to this solution, mixed thoroughly, and then heated at 37°C to prepare the test solution. Alternatively, a pH 7.0 potassium phosphate / sodium hydroxide buffer (0.1 mol / L) or water was used in place of the sample solution to prepare the comparative solution. The absorbance of the comparative solution and the test solution at a wavelength of 500 nm was measured 2 and 5 minutes after the addition of the sample solution. The amount of glucose oxidized was quantified based on the molar absorption coefficient of the generated quinoneimine dye, with 1 unit defined as the amount of enzyme required to oxidize 1 μmol of glucose in 1 minute.

[0069] In the present embodiment, when a glyceride-containing composition is used as a substrate, a lipase can be used in conjunction with the manufacturing process of the processed unsaturated fatty acid composition. As long as the lipase shows the effect of hydrolyzing glycerides to free fatty acids, its type and source are not particularly limited. Examples of lipases include, for example, lipases derived from the genus Rhizopus, the genus Aspergillus, the genus Mucor, the genus Rhizomucor, the genus Thermomyces, the genus Pseudomonas, the genus Geotrichum; lipases derived from the genus Penicillium, the genus Candida, etc. Preferably, a lipase derived from Candida rugosa can be cited.

[0070] The amount of lipase used is not particularly limited. The amount of lipase added per 1 mg of glyceride is preferably, for example, 0.1 U or more, 0.5 U or more, 1 U or more, 5 U or more, 10 U or more, 20 U or more, 30 U or more, or 40 U or more. Furthermore, the amount of lipase added per 1 mg of glyceride is preferably, for example, 5000 U or less, 1000 U or less, 500 U or less, 200 U or less, 100 U or less, 90 U or less, 80 U or less, or 70 U or less.

[0071] <Method for preparing fatty acid α-hydroxylase>

[0072] Methods for preparing fatty acid α-hydroxylase include recovering fatty acid α-hydroxylase from the culture fluid or cells of a microorganism or a recombinant microorganism into which the gene for the enzyme has been introduced. For example, when using a microorganism that secretes fatty acid α-hydroxylase, the cells can be recovered from the culture fluid by filtration, centrifugation, or the like, as needed, and then the enzyme can be isolated and / or purified. Alternatively, when using a microorganism that does not secrete fatty acid α-hydroxylase, the cells can be recovered from the culture fluid as needed, crushed by pressure treatment, ultrasonic treatment, or the like to expose the enzyme, and then the enzyme can be isolated and / or purified. Known protein separation and / or purification methods can be used without particular limitation as methods for isolating and / or purifying the enzyme, including centrifugation, UF concentration, salting-out, and various chromatography methods using ion exchange resins. The purified fatty acid α-hydroxylase can be powdered by freeze-drying, vacuum drying, or spray drying, as needed.

[0073] <Production Conditions for Processed Unsaturated Fatty Acid-Containing Composition>

[0074] The reaction time, temperature, pH of the reaction solution, etc. in the process of allowing fatty acid α-hydroxylase to act on the composition containing unsaturated long-chain fatty acids are not particularly limited. The reaction temperature is, for example, 10 to 70°C, preferably 20 to 60°C, and more preferably 30 to 50°C. The pH of the reaction solution is, for example, 4 to 10, preferably 5 to 9, and more preferably 6 to 8. The reaction time is, for example, 30 seconds to 24 hours, preferably 1 minute to 12 hours, more preferably 2 minutes to 6 hours, 3 minutes to 2 hours, 4 minutes to 1 hour, and 5 minutes to 30 minutes. The above reaction conditions can be used to more effectively shorten the unsaturated long-chain fatty acids. These reaction conditions are appropriately selected according to the target processed unsaturated fatty acid composition. It should be noted that the optimal reaction conditions can be determined by preliminary experiments.

[0075] By using the production method of this embodiment, a composition containing a processed unsaturated fatty acid with a shortened (shortened) chain length can be produced. One embodiment of the production method of the composition containing a processed unsaturated fatty acid of this embodiment includes the following steps (1) and (2).

[0076] Step (1): Preparing a raw material containing unsaturated long-chain fatty acids (composition containing unsaturated long-chain fatty acids)

[0077] Step (2): Treat the prepared raw materials with fatty acid α-hydroxylase

[0078] It should be noted that after step (2), a step (3) of inactivating the enzyme and / or a step (4) of recovering the short-chained unsaturated fatty acids may be added. In addition, step (2) may be a step of treating in the presence of hydrogen peroxide.

[0079] Another aspect of the production method of this embodiment includes the following steps (1) and (2).

[0080] Step (1): Preparing a raw material containing unsaturated long-chain fatty acids (composition containing unsaturated long-chain fatty acids)

[0081] Step (2): Treat the prepared raw material with fatty acid α-hydroxylase in the presence of an oxidase substrate and an oxidase.

[0082] Note that, after step (2), a step (3) of inactivating the enzyme and / or a step (4) of recovering the short-chained processed unsaturated fatty acids may be added.

[0083] Another aspect of the production method of this embodiment includes the following steps (1) to (3).

[0084] Step (1): Preparing a raw material containing glycerides to which unsaturated long-chain fatty acids are bonded (composition containing unsaturated long-chain fatty acids)

[0085] Step (2): Treat the prepared raw materials with lipase

[0086] Step (3): Treat the lipase-treated raw material with fatty acid α-hydroxylase

[0087] It should be noted that after step (3), a step (4) of inactivating the enzyme and / or a step (5) of recovering the saturated fatty acids after shortening the chain may be added. In addition, step (3) may be a step of treating the raw material treated with lipase with fatty acid α-hydroxylase in the presence of hydrogen peroxide, or a step of treating the raw material treated with oxidase and fatty acid α-hydroxylase in the presence of a substrate for the oxidase.

[0088] (Contains processed unsaturated fatty acid composition)

[0089] The present embodiment can also relate to the above-mentioned manufacture method containing the processing unsaturated fatty acid composition and manufacture containing the processing unsaturated fatty acid composition.Containing the processing unsaturated fatty acid composition comprises the processing unsaturated fatty acid (short-chain unsaturated fatty acid) that the unsaturated long-chain fatty acid short-chain is obtained.That is, compared with the carbon chain length of unsaturated long-chain fatty acid, the carbon chain length of processing unsaturated fatty acid shortens.In addition, containing the processing unsaturated fatty acid composition preferably contains odd number unsaturated fatty acids (C17:1, C15:1, C13:1, C11:1, C9:1, C7:1, C5:1, C3:1).

[0090] The processed unsaturated fatty acid composition can include any component contained in the unsaturated long-chain fatty acid composition. In addition, the form of the processed unsaturated fatty acid composition can be any form of powder, solid, gel, or liquid (paste). Among them, the processed unsaturated fatty acid composition is preferably in liquid (paste).

[0091] In the present embodiment, the processed unsaturated fatty acid composition can be a processed unsaturated fatty acid food. In addition, the unsaturated long-chain fatty acid composition can also be a unsaturated long-chain fatty acid food. The unsaturated long-chain fatty acid food is a food containing an unsaturated long-chain fatty acid, a compound having an unsaturated long-chain fatty acid in its partial structure, and as such food, for example, processed oils and fats (olive oil, safflower oil, sunflower seed oil, rapeseed oil, peanut oil and rice bran oil, etc.), fatty acid-containing foods (meat products, dairy products (cheese, processed milk, nut milk, yogurt), egg products, etc.) can be enumerated.

[0092] (Enzyme)

[0093] This embodiment relates to an enzyme agent for shortening the chain length of unsaturated long-chain fatty acids, containing a fatty acid α-hydroxylase. The enzyme agent for shortening the chain length of unsaturated long-chain fatty acids contains the aforementioned fatty acid α-hydroxylase as an active ingredient. It should be noted that the enzyme agent for shortening the chain length of unsaturated long-chain fatty acids can be composed of the aforementioned fatty acid α-hydroxylase. Examples of the fatty acid α-hydroxylase include the aforementioned fatty acid α-hydroxylase, and the preferred range is the same.

[0094] The content of the fatty acid α-hydroxylase in the enzyme agent of the present embodiment is not particularly limited and can be appropriately set within a range in which the catalytic activity of shortening the chain length of unsaturated fatty acids can be exhibited.

[0095] The enzyme agent of this embodiment may contain other components in addition to the fatty acid α-hydroxylase to the extent that the effects of the present invention are not affected. Examples of other components include enzymes other than the fatty acid α-hydroxylase, additives, and culture residues produced during the steps of preparing the fatty acid α-hydroxylase. Preferably, the enzyme agent contains other enzymes (such as oxidase) in addition to the fatty acid α-hydroxylase.

[0096] When the enzyme agent of the present embodiment contains an oxidase, the oxidase is preferably an enzyme that generates hydrogen peroxide by reacting with an oxidized substrate. Examples of oxidases include sugar oxidases with a wide range of sugars including glucose as substrates, glucose oxidases with glucose as substrates, sugar alcohol oxidases with glycerol as substrates, and oxalate oxidases with oxalic acid as substrates. Among them, the oxidase is preferably a sugar oxidase. The type of sugar oxidase is not particularly limited. For example, sugar oxidases derived from Acremonium chrysogenum can be mentioned. When using an oxidase, the enzyme agent preferably contains a substrate as needed. In addition, the enzyme agent can be a single dose or two or more doses.

[0097] In addition, other enzymes that can be included in the enzyme agent of the present embodiment include amylase (α-amylase, β-amylase, glucoamylase), glucosidase (α-glucosidase, β-glucosidase), galactosidase (α-galactosidase, β-galactosidase), protease (acid protease, neutral protease, alkaline protease), peptidase (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatase (acid phosphatase, alkaline phosphatase), nuclease, deaminase, oxidase, dehydrogenase, glutaminase, pectinase, catalase, glucanase, transglutaminase, protein deamidase, pullulanase, peroxidase, superoxide dismutase, etc. These other enzymes can be one or more.

[0098] As additives that can be contained in enzyme agents, excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline, etc. can be enumerated. As excipients, starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol, etc. can be enumerated. As buffers, phosphates, citrates, acetates, etc. can be enumerated. As stabilizers, propylene glycol, ascorbic acid, etc. can be enumerated. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methyl parahydroxybenzoate, etc. can be enumerated. As antiseptics, ethanol, benzalkonium chloride, p-hydroxybenzoic acid, chlorobutanol, etc. can be enumerated. These additives can be one or more.

[0099] The form of the enzyme agent of the present embodiment is not particularly limited, and examples thereof include powder, solid, gel, liquid (paste), and immobilized on a carrier.

[0100] (Method for shortening the chain length of unsaturated long-chain fatty acids / Method for producing foods containing processed unsaturated fatty acids)

[0101] This embodiment may also relate to a method for shortening the chain length of unsaturated long-chain fatty acids, comprising the steps of: allowing a fatty acid α-hydroxylase to act on an unsaturated long-chain fatty acid or a compound partially containing an unsaturated long-chain fatty acid. By allowing the fatty acid α-hydroxylase to act on the unsaturated long-chain fatty acid or the compound partially containing an unsaturated long-chain fatty acid, a shortened unsaturated fatty acid is obtained.

[0102] In addition, the present embodiment may also relate to a method for producing a food containing processed unsaturated fatty acids, comprising the steps of allowing a fatty acid α-hydroxylase to act on a food containing unsaturated long-chain fatty acids or a food containing a compound having an unsaturated long-chain fatty acid as part of its structure. The above-mentioned food is a food or food material containing an unsaturated long-chain fatty acid or a compound having an unsaturated long-chain fatty acid as part of its structure. Examples of such food or food materials include edible oils (olive oil, safflower oil, sunflower oil, rapeseed oil, peanut oil, and rice bran oil), meat (beef, chicken, and pork), cheese, nuts (macadamia nuts, almonds, etc.), peanuts, sunflower seeds, eggs, milk, olives, and avocados.

[0103] Example

[0104] Below, give embodiment and feature of the present invention and be described in more detail.As long as the material, usage amount, ratio, processing content, processing sequence etc. shown in the following embodiment do not depart from the gist of the present invention, then can suitably change.Therefore, the scope of the present invention should not be interpreted as the specific example shown below by restrictiveness.

[0105] (Materials used)

[0106] Enzymes: The enzymes used were described below.

[0107] Fatty acid α-hydroxylase: Cytochrome P450 from Exiguobacterium AT1b (P450Exα)

[0108] Carbohydrate oxidase: Carbohydrate oxidase derived from Cephalosporium acremonium (manufactured by Amano Enzyme Co., Ltd.)

[0109] [Table 1]

[0110] Unsaturated fatty acids Abbreviation Examples of foods containing Manufacturer content Oleic acid C18:1 Sunflower oil, olive oil, rapeseed oil Tokyo Chemical Industry >99.0％ Palmitoleic acid C16:1 Macadamia nuts, beef Cayman Chemical ≥99％ Myristic acid C14:1 beef Cayman Chemical ≥95％ saturated fatty acids Abbreviation Examples of foods containing large amounts Manufacturer content stearic acid C18 Beef, pork, chocolate Tokyo Chemical Industry >98％ Palmitic acid C16 Palm oil, butter, tallow Wako 95％ Myristic acid C14 Coconut oil, coconut, butter Wako 98％

[0111] (Method for obtaining enzyme)

[0112] The gene for the synthetic fatty acid peroxidase derived from Exiguobacterium AT1b (P450Exα gene: sequence number 2) was amplified by PCR. The amplified DNA was inserted into an Escherichia coli expression vector (pET28a) and introduced into the E. coli BL21 strain by chemical transformation. The resulting transformant was inoculated into a liquid culture medium (pH 7.0) containing yeast extract, peptone, and glycerol, and cultured at 25°C with aeration and stirring for 24 hours. After the bacteria were recovered from the culture solution, they were crushed using a beads oscillator (beads shocker) to extract the enzyme. The resulting extract was concentrated by ultrafiltration and purified by an affinity column to obtain an enzyme solution.

[0113] (Method for measuring enzyme concentration)

[0114] The concentration of the enzyme (P450Exα) in the enzyme solution is measured using the pyridine hematin method. First, pyridine is added to a sodium hydroxide solution to a final concentration of 20mM, and an arbitrary amount of enzyme solution is added thereto and mixed. This releases the heme b in P450Exα. Sodium dithionite is added thereto to reduce the heme b. Pyridine coordinates with the heme b that has reached the reduced state, and absorption at a characteristic wavelength of 557nm is observed. Then, based on the molar absorption coefficient of 34.7mM －1 cm －1 The concentration of P450Exα in the enzyme solution was calculated.

[0115] (Method for measuring P450Exα enzyme activity)

[0116] Add 0.1M potassium phosphate, 0.5M potassium acetate, and 0.5mM hydrogen peroxide to a plastic cuvette and incubate at 40°C for 30 minutes. Then, add 5mM 2,6-dimethoxyphenol and incubate at 40°C for 1 minute. Next, add an enzyme solution diluted to an appropriate concentration and measure the absorbance change at 469nm using a spectrophotometer over a 3-minute period. Calculate the difference in absorbance between 130 and 150 seconds to determine the reaction rate. 1 unit (1U) is calculated as the amount of enzyme required to oxidize 1 nmol of substrate in 1 minute.

[0117] (Example 1)

[0118] (1) Fatty acid shortening reaction based on P450Exα

[0119] (method)

[0120] Potassium phosphate buffer (pH 7) (reaction concentration 0.1 M), substrate (oleic acid: reaction concentration 100 μM), and P450Exα (reaction concentration 1080 U / mL) were added to a 6cc vial to a reaction volume of 0.5 mL and incubated at 40°C for 1 minute. Hydrogen peroxide was then added to a reaction concentration of 10 mM, and the reaction was continued at 40°C. The substrate was dissolved in ethanol, and the final ethanol concentration in the reaction solution was 1%. Reaction times were 0, 1, 5, 10, and 20 minutes. After each reaction time, 1 mL of chloroform and 5 μL of 6N HCl were added to 0.5 mL of the reaction solution, stirred thoroughly, and 600 μL of the chloroform layer was aliquoted into a new vial and air-dried overnight. After the solvent was distilled off, 300 μL of BSTFA-TMCS (99:1) (Tokyo Chemical Industry Co., Ltd.) was added to the vial and allowed to stand for at least 1 hour. 1 μL of this solution was injected into a GC-MS to determine the abundance ratio of the fatty acids after the reaction. The abundance ratio was calculated by calculating the ratio of each fatty acid peak to the sum of the peaks corresponding to m / z = 117 (fatty acid fragment) in the GC-MS.

[0121] GCMS analysis conditions

[0122]

[0123] The fatty acid abundance ratios at each reaction time when oleic acid (C18:1) was used as a substrate are shown below.

[0124] [Table 2]

[0125]

[0126] (Example 2)

[0127] A fatty acid shortening reaction was performed using P450Exα in the same manner as in Example 1, except that the substrate was changed to palmitoleic acid. The fatty acid abundance ratios at each reaction time when palmitoleic acid (C16:1) was used as the substrate are shown below.

[0128] [Table 3]

[0129]

[0130] (Example 3)

[0131] A fatty acid shortening reaction was performed using P450Exα in the same manner as in Example 1, except that the substrate was changed to myristoleic acid. The fatty acid abundance ratios at each reaction time when myristoleic acid (C14:1) was used as the substrate are shown below.

[0132] [Table 4]

[0133]

[0134] (Comparative Example 1)

[0135] A fatty acid shortening reaction was carried out using P450Exα in the same manner as in Example 1, except that the substrate was changed to stearic acid. The fatty acid abundance ratios at each reaction time when stearic acid (C18) was used as the substrate are shown below.

[0136] [Table 5]

[0137]

[0138] (Comparative Example 2)

[0139] A fatty acid shortening reaction was carried out using P450Exα in the same manner as in Example 1, except that the substrate was changed to palmitic acid. The fatty acid abundance ratios at each reaction time when palmitic acid (C16) was used as the substrate are shown below.

[0140] [Table 6]

[0141]

[0142] (Reference Example 1)

[0143] A fatty acid shortening reaction was carried out using P450Exα in the same manner as in Example 1, except that the substrate was changed to myristic acid. The fatty acid abundance ratios at each reaction time when myristic acid (C14) was used as the substrate are shown below.

[0144] [Table 7]

[0145]

[0146] (result)

[0147] According to the above results, it is shown that P450Exα has a fatty acid shortening effect on any fatty acid, but when using saturated fatty acids as substrates, there is a trend of reduced reactivity and residual substrate in long-chain fatty acids (C16, C18) with more than 16 carbon atoms. On the other hand, when using unsaturated fatty acids as substrates, no matter how long the fatty acid chain is, most of the substrates are consumed after 10 minutes of the reaction start and converted into short-chain fatty acids. It was originally believed that unsaturated fatty acids would be hindered from contacting with enzymes due to the rigid double bonds in their structure, but contrary to expectations, it was found that they were more reactive than saturated fatty acids. In addition, it was found that according to the present invention, odd-numbered unsaturated fatty acids (C17:1, C15:1, C13:1, C11:1, C9:1) that are almost non-existent in nature and difficult to synthesize can be generated.

[0148] (Example 4)

[0149] (2) Fatty acid shortening reaction based on the combined use of P450Exα and oxidase

[0150] (method)

[0151] Potassium phosphate buffer (pH 7) (reaction concentration 0.1 M) and substrate (oleic acid: reaction concentration 500 μM) were added to a 6cc vial to a reaction volume of 0.5 mL. The solution was then allowed to stand at 40°C for 5 minutes. P450Exα (reaction concentration 1080 U / mL) was added, and the mixture was incubated at 40°C for 1 minute. Hydrogen peroxide (reaction concentration 10 mM) was then added and the reaction continued at 40°C for 1 hour. The substrate was dissolved in ethanol, and the final ethanol concentration in the reaction solution was 1%. After each reaction time, 1 mL of chloroform and 5 μL of 6N HCl were added to 0.5 mL of the reaction solution, and the mixture was stirred thoroughly. 300 μL of the chloroform layer was collected in a new vial, and 300 μL of BSTFA-TMCS (99:1) (Tokyo Chemical Industry) was added to the chloroform layer. The mixture was allowed to stand for at least 1 hour. 1 μL of this solution was injected into a GC-MS and analyzed in the same manner as above to calculate the fatty acid abundance ratio.

[0152] (Examples 5 and 6)

[0153] Potassium phosphate buffer (pH 7) (reaction concentration 0.1 M), glucose (reaction concentration 0.2 mass %), and substrate (oleic acid: reaction concentration 500 μM) were added to a 6cc vial to a reaction volume of 0.5 mL. The solution was then allowed to stand at 40°C for 5 minutes. P450Exα (reaction concentration 1080 U / mL) and carbohydrate oxidase (reaction concentration 1.5 U / mL or 0.75 U / mL) were then added, and the reaction was allowed to proceed at 40°C for 1 hour. The substrate was dissolved in ethanol, and the final ethanol concentration in the reaction solution was 1%. After each reaction time, 1 mL of chloroform and 5 μL of 6N HCl were added to 0.5 mL of the reaction solution. The solution was stirred thoroughly, and 300 μL of the chloroform layer was collected in a new vial. 300 μL of BSTFA-TMCS (99:1) (Tokyo Chemical Industry Co., Ltd.) was added to the chloroform layer, and the solution was allowed to stand for at least 1 hour. 1 μL of this solution was injected into GC-MS and analyzed in the same manner as above to calculate the abundance ratio of fatty acids.

[0154] (result)

[0155] The results are shown below. They indicate that the same effect can be obtained by adding oxidase and its substrate instead of hydrogen peroxide.

[0156] [Table 8]

[0157] Example 4 Example 5 Example 6 P450Exα(U / mL) 1080 1080 1080 Hydrogen peroxide (mM) 10 - - Carbohydrate oxidase (U / mL) - 0.75 1.5 glucose(%) - 0.2 0.2 C13:1 11 12 13 C14:1 37 37 33 C15:1 23 28 24 C16:1 12 16 13 C17:1 0 0 0 C18:1 17 7 17

[0158] Industrial applicability

[0159] According to the present invention, short-chain unsaturated fatty acids can be produced while maintaining the double bond portion of the unsaturated fatty acids. Furthermore, according to the present invention, compositions containing short-chain unsaturated fatty acids can be obtained. The present invention provides a technology for altering the chain length (shortening) of unsaturated fatty acids, which is particularly useful in the pharmaceutical and food fields.

[0160] SEQ ID NO: 1: Amino acid sequence of cytochrome P450 (P450Exα) derived from Exiguobacterium AT1b

[0161] SEQ ID NO: 2: Base sequence of the cytochrome P450 (P450Exα) gene derived from Exiguobacterium AT1b

[0162] Serial Number 1

[0163] MGKVIPKQEGLDHSVDFLREGYLFVANRRKSFQSNIFESRLLGERVICLGGEEAAEVFYDANKFTRQDAAPKRLLKTLFGEGGVQTLDGSEHTHRKQMFMSLMTKENIDRLLRLTYREWNQIERMGEEIVLYDIAQEVLMKAVCEWSGVPLAKEEVGKRTEEMRLLFESGTSLGPTYLQGRKARSSAEVWIRQMVKEVRSNRLLPN EHTALYEFSWHRDESGELLPEEVVAVEVLNILRPTVAISVYVLFTVLALHQFPDVKEQVERGEVSKTEVVQEVRRFYPFFFPVAAARVKTDFEWDGYAFPEGTLTLLDLYGTNHDVSIWTEPDRFDPSRFKDWKESPFNIPQGGGDVDFGHRCAGEHVTIAILAQVIELFTKEYAYTVPPQDLSYSFVDMPSLPKSKLRLTHLTRNQ

[0164] serial number 2

[0165]

Claims

1. A method for producing a processed unsaturated fatty acid composition, comprising the steps of: allowing a fatty acid α-hydroxylase to act on a composition containing an unsaturated long-chain fatty acid or a composition containing a compound having an unsaturated long-chain fatty acid as a partial structure.

2. The method for producing a processed unsaturated fatty acid-containing composition according to claim 1, wherein The fatty acid α-hydroxysuccinyltransferase is cytochrome P450.

3. The method for producing a processed unsaturated fatty acid-containing composition according to claim 1, wherein The fatty acid α-hydroxysuccinyltransferase is composed of the amino acid sequence of sequence number 1 or an amino acid sequence equivalent thereto.

4. The method for producing a processed unsaturated fatty acid-containing composition according to claim 1, wherein The unsaturated long-chain fatty acid composition contains at least one selected from the group consisting of myristic acid, palmitic acid and oleic acid.

5. The method for producing a processed unsaturated fatty acid-containing composition according to claim 1, wherein The composition containing a compound having unsaturated long-chain fatty acids in a partial structure is a glyceride-containing composition.

6. A method for shortening the chain length of unsaturated long-chain fatty acids, comprising the following steps: allowing fatty acid α-hydroxylase to act on unsaturated long-chain fatty acids or compounds having unsaturated long-chain fatty acids in part of their structure.

7. A method for producing a food containing processed unsaturated fatty acids, comprising the steps of: allowing fatty acid α-hydroxylase to act on a food containing unsaturated long-chain fatty acids or a food containing a compound having unsaturated long-chain fatty acids in part of its structure.

8. An enzyme for shortening the chain length of unsaturated long-chain fatty acids, comprising fatty acid α-hydroxylase.

9. The enzyme agent for shortening the chain length of unsaturated long-chain fatty acids according to claim 8, wherein The fatty acid α-hydroxysuccinyltransferase is cytochrome P450.

10. The enzyme agent for shortening the chain length of unsaturated long-chain fatty acids according to claim 8, wherein The fatty acid α-hydroxysuccinyltransferase is composed of the amino acid sequence of sequence number 1 or an amino acid sequence equivalent thereto.

11. The enzyme agent for shortening the chain length of unsaturated long-chain fatty acids according to claim 8, wherein The unsaturated long-chain fatty acid is at least one selected from the group consisting of myristic acid, palmitic acid and oleic acid.

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