Method for producing, in bioreactor, resolvin d5 from docosahexaenoic acid-enriched oil by using lipoxygenase

Abstract

The present invention relates to a composition and a method for preparing resolvin D5, which is a human endogenous substance, in a flask and a bioreactor through whole-cell reactions by using dual 15-lipoxygenase derived from bacterial strain Archangium violaceum with, as substrates, a high-purity docosahexaenoic acid reagent and polyunsaturated fatty acids obtained through solvent extraction after hydrolysis from low-cost docosahexaenoic acid-enriched oil, and, more specifically, to: a method and a preparation composition therefor, the method being for obtaining docosahexaenoic acid having a purity of 90% through a purification process of using commercial lipase and adsorbent resins to effectively hydrolyze docosahexaenoic acid-enriched oil into polyunsaturated fatty acids, which include docosahexaenoic acid, and then performing solvent extraction thereon; and a method and a preparation composition therefor, the method being for mass-producing resolvin D5, which is a human endogenous substance, in a flask and a bioreactor through whole-cell reactions by using bacteria Archangium violaceum-derived dual arachidonic acid 15-lipoxygenase with, as substrates, a purified hydrolysate and a high-purity docosahexaenoic acid reagent. According to the present invention, the composition can be produced with high productivity and high yield by means of an eco-friendly method, and can be effectively used in various industrial fields such those of medicine, food and cosmetics.

Description

Method for producing resolvin D5 from oil enriched with docosahexaenoic acid by lipooxidizing enzyme in a bioreactor

[0001] The present invention relates to a composition for producing high-purity resolvin D5 by whole-cell reaction of a recombinant microorganism containing double-oxygenating arachidonate 15-lipoxygenase derived from a strain of Archangium violaceum in a bioreactor, and a method for producing resolvin D5, an endogenous substance of the human body, using a hydrolyzed extract of oil containing DHA derived from inexpensive docosahexaenoic acid-fortified fish (e.g., tuna) or microalgae as a substrate.

[0002] This invention was carried out with the support of the research project "Identification of Microbial Biosynthesis and Inflammation / Obesity Regulation Mechanism of Human Oxylipin Based on Metabolite Profiling" (Project Identification Number: 1711154866, Implementing Organization: Konkuk University) of the Group Research Support Project supported by the Ministry of Science and ICT.

[0003] Dihydroxy fatty acids (DiHFAs) with 20 to 22 carbon atoms are lipid regulators with two hydroxyl groups. They are mainly present in lipids of animals and humans and are substances produced from lipids. They are biosynthesized in vivo by the combined reaction of cyclooxygenase (COX), cytochrome P450 (CYP), and lipoxygenase (LOX) that have been processed with aspirin from animal unsaturated fatty acids. Dihydroxy fatty acids produced from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are lipid regulators and belong to the specialized pro-resolving mediators (SPMs), a type of human signaling molecule. SPMs are important substances involved in various physiological functions such as microbial elimination, pain relief, tissue regeneration, anti-inflammatory response, and homeostasis regulation in animals, including humans, as endogenous regulators of infection and inflammation. Pro-inflammatory mediators include leukotrienes (LT) and prostaglandins (PG), and anti-inflammatory mediators include protectins (PT), resolvins (RV), maresins (MaR), and lipoxins (LX). Among them, resolvins are endogenous lipid mediators produced by the combined reaction of lipolytic enzymes and double oxidation reactions. They are 20 or 22 carbon omega fatty acids derived from eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0004] Resolvin (RV) is a mediator that promotes inflammation resolution. It is mainly converted into resolvin E and resolvin D by the action of lipooxidase on animal polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid, respectively. Resolvin is known as a substance that fundamentally treats inflammation and infection by promoting phagocytosis and removing viruses, bacteria, and inflammation, and restoring damaged tissues. Among them, resolvin D5 (RVD5) can treat inflammation even at extremely small levels in the human body, reduce pain, reduce the use of antibiotics in infection treatment, and has functions such as skin regeneration. In addition, it has been confirmed that it exhibits anti-inflammatory activity by reducing inflammation caused by intracellular oxidative stress, and prevents degeneration by inhibiting cell death through phagocytosis. Accordingly, mass production of the ultra-expensive anti-inflammatory mediator, Resolvin D5 (approximately 17 million won / mg), from low-cost docosahexaenoic acid-enriched oil (80% (w / w) 200,000 won / kg) through production optimization would facilitate industrialization. Furthermore, Resolvin D5 suggests that it can be practically applied to functional and efficacy studies in academic research in medicine and biotechnology as a next-generation therapeutic material capable of resolving inflammation and infection.

[0005] Lipoxygenase (LOX) catalyzes the stereoselective and regioselective peroxidation of polyunsaturated fatty acids containing one or more cis, cis-1,4-pentadiene units through a dioxygenating reaction. Stereospecific lipoxygenases are catalyzed by chirality or handedness, with R- or S-forms being the preferred configurations. These are called R-lipoxygenases and S-lipoxygenases, respectively. The positional specificity is different depending on the carbon length of the polyunsaturated fatty acid used as a substrate and the position of the pentadiene structure. Among them, hydroxyl groups are generated at carbon positions 5, 8, 9, 11, 12, and 15 of arachidonic acid, an animal polyunsaturated fatty acid, and this enzyme generates hydroxyl groups at carbon positions 7, 10, 11, 13, 14, and 17 in polyunsaturated fatty acids with 22 carbons.

[0006] In the case of double lipooxidase, it is known that hydroperoxide is generated at two carbon positions, and the cis-cis form is generally transformed into a cis-trans form structure while forming a hydroxyl group through a catalytic reaction. The double lipooxidases known so far are mouse double dioxygenating ARA 8-LOX, Sphingpyxis macrogoltabida double arachidonic acid 9-LOX, Endozoicomonas numazuensis double arachidonic acid 12-LOX, and Archangium violaceum double arachidonic acid 15-LOX. In mammals, dihydroxylated fatty acids formed by dual 8-lipooxidase are biosynthesized in vivo by intracellular responses to tissue damage and infection, and are biosynthesized in small amounts of resolvin D5 at less than 250 pg / mL. It has been reported that the production of dihydroxylated fatty acids by three types of bacterial lipooxidases using whole cells was performed in a single-step bioconversion. Of these, the most active and pro-inflammatory mediator is dual 15-lipooxidase from Arcangium violacium, but it has not been produced at the g / L level to date, and the production of pro-inflammatory mediators in a bioreactor using a whole-cell reaction has not been reported. Dual arachidonic acid 15-lipooxidase from Arcangium violacium selectively forms S-hydroxy groups at the 5th and 15th carbon positions in 20-C unsaturated fatty acids such as arachidonic acid. Additionally, in unsaturated fatty acids with 22 carbon atoms, S-hydroxyl groups are formed at the 7th and 17th carbon positions.

[0007] The synthetic method of resolvin D5 reported to date has limitations in that it is synthesized with a yield of about 20% through a 25-step chemical synthesis method, and benzene analogs and heavy metals are used and released during the synthetic process, causing environmental pollution. In addition, a previous report reported that resolvin D5 was biosynthesized at a level of about 0.1 g / L by dual arachidonic acid 15-oxidation lipase from Arcangium violacium using whole cells. However, in bioconversion using whole cells, the oxidation reaction was inhibited by a certain concentration of substrate and produced intermediate, and it is known that the inhibition effect of hydrophobic fatty acids is overcome by sufficient oxygen supply and solvent use during the catalytic reaction.

[0008] In the present invention, docosahexaenoic acid-enriched oil was effectively hydrolyzed into polyunsaturated fatty acids including docosahexaenoic acid using commercial lipase and adsorption resin to secure a substrate, and the hydrolyzate was converted into resolvin D5, an endogenous human substance, through whole-cell reaction in a flask and bioreactor using dual arachidonic acid 15-lipooxidase from the bacterium Arcangium violacium as a substrate. As a result, resolvin D5 was biosynthesized in g / L units with a high yield of 70% or higher and a purification yield of 97% or higher. In addition, maximum conversion was confirmed using a high-purity 95, 99% docosahexaenoic acid reagent as a substrate. To date, the optimization and production of resolvin D5 in bioreactors and flasks using polyunsaturated fatty acids hydrolyzed from high-purity docosahexaenoic acid reagents and low-cost docosahexaenoic acid enriched oil as substrates for the production of resolvin D5 has not been reported.

[0009] The present invention was derived from the above-mentioned necessity, and the purpose of the present invention is to provide a composition for producing a dihydroxy fatty acid having 20 to 22 carbon atoms, which contains a polyunsaturated fatty acid derived from oil containing DHA obtained from fish or microalgae at a high purity.

[0010] The present invention provides a composition for producing a dihydroxy fatty acid having 20 to 22 carbon atoms, comprising a lipase that hydrolyzes oil containing DHA obtained from inexpensive fish or microalgae into a polyunsaturated fatty acid (containing docosahexaenoic acid in high purity) and an adsorbent resin.

[0011] The present invention also aims to provide a method for purifying high-purity (about 70 to 99%) docosahexaenoic acid by using an additional solvent extraction method on the hydrolyzate (polyunsaturated fatty acid including docosahexaenoic acid) produced by the hydrolysis. In the solvent extraction of the present invention, it is preferable to extract with a solvent in which hexane and acetonitrile are mixed in a volume ratio of 3 to 5:1 (more preferably 4:1), and any extraction method within the field of the present invention can be used without limitation.

[0012] The present invention also provides a method for producing resolvin D5 through bioconversion using a composition containing the high-purity docosahexaenoic acid (a hydrolysate purified in a flask and a bioreactor through a whole-cell reaction) as a substrate. The present invention aims to provide a composition for producing resolvin D5, an endogenous substance in the human body, using the substrate and a dual 15-lipolytic enzyme derived from a strain of Archangium violaceum, and a method for producing resolvin D5.

[0013] Specifically, the present invention provides a method for producing resolvin D5, an endogenous substance of the human body, by treating a microorganism transformed with a dual 15-oxidation lipase gene derived from a strain of Archangium violaceum with a high-purity 95, 99% docosahexaenoic acid reagent (obtained in a flask and bioreactor through a whole-cell reaction) as a substrate.

[0014] Accordingly, the present invention provides a composition of high-purity polyunsaturated fatty acids (including docosahexaenoic acid) obtained by hydrolyzing oil obtained from fish or microalgae with commercial lipase and an adsorption resin and then refining the same, a composition for producing resolvin D5 in high yield through a bioconversion process using the composition containing the high-purity polyunsaturated fatty acid as a substrate, a method for producing resolvin D5 using the substrate, and resolvin D5 produced by the method.

[0015] Another object of the present invention is to provide a composition for producing dihydroxy fatty acid or resolvin D5, which contains the enzyme as an active ingredient.

[0016] Another object of the present invention is to provide a method for obtaining a polyunsaturated fatty acid as a substrate from oil obtained from fish or microalgae and using the same to produce dihydroxy fatty acid or resolvin D5.

[0017] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0018] The terminology used herein is for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" should be understood to indicate the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but do not preclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof. Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning they have in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined in this application.

[0019] It is already known that resolvin D5 can be used as a research material in various fields such as medicine, biology, and biotechnology. However, a method for biologically producing resolvin D5 in a high yield of 70% or higher at a g / L level using polyunsaturated fatty acids obtained by hydrolysis of high-purity docosahexaenoic acid or inexpensive docosahexaenoic acid-enriched oil in a flask or bioreactor has not been developed. Furthermore, a method for purifying docosahexaenoic acid with a purity of 97% or higher from oil obtained from fish or microalgae has not been developed.

[0020] To achieve the above-described object, the present inventors used commercial lipase and an adsorption resin to hydrolyze oil (docosahexaenoic acid-enriched oil) obtainable from inexpensive fish or microalgae into polyunsaturated fatty acids containing 70% or more (about 70% to 80% or 70% to 99%) docosahexaenoic acid, and then secured high-purity (about 90%) docosahexaenoic acid through a solvent extraction method. Using the composition containing the obtained high-purity docosahexaenoic acid as a substrate, the present inventors conducted continuous research to more effectively produce resolvin D5, a dihydroxy fatty acid, through an optimized bioconversion process. Specifically, the present inventors obtained docosahexaenoic acid, which serves as a substrate in a bioconversion process, by separating and purifying it to a purity of 90% from docosahexaenoic acid-enriched oil, and produced the final product, resolvin D5 (7S,17S-dihydroxy-4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid), through an eco-friendly bioconversion method using a composition containing purified, high-purity docosahexaenoic acid as a substrate and a dual 15-lipooxidase derived from the Archangium violaceum strain through a whole-cell reaction in a flask and a bioreactor. Thereafter, by treating a solvent and a polymer in the bioconversion process through optimization, the solubility of the substrate was efficiently increased, and it was confirmed that sufficient oxygen supply in the bioreactor could be used to manufacture it with high productivity and high yield, thereby completing the present invention.

[0021] Accordingly, the present invention provides a composition for producing a dihydroxy fatty acid having 20 to 22 carbon atoms, which contains a polyunsaturated fatty acid derived from DHA-containing oil obtained from fish or microalgae, etc., as a substrate, with high purity.

[0022] In the above composition, the polyunsaturated fatty acid derived from the DHA-containing oil may include docosahexaenoic acid (DHA).

[0023] The polyunsaturated fatty acid obtained after hydrolysis of the above composition may contain docosahexaenoic acid in high purity.

[0024] In this specification, “oil” is a concept that includes all oils containing DHA derived from microalgae, fungi, fish, etc.

[0025] The term "dihydroxy fatty acid" as used herein refers to a fatty acid, preferably an unsaturated fatty acid having 20 to 22 carbon atoms, in which two hydroxyl groups are substituted.

[0026] In the present specification, the dihydroxy fatty acid having 20 to 22 carbon atoms includes resolvin D5.

[0027] In this specification, resolvin D5 is a compound represented by the following chemical formula 1, and its name is 7S,17S-dihydroxy-4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid, and is also called 7S,17S-dihydroxydocosahexaenoic acid (DiHDHA).

[0028] [Chemical Formula 1]

[0029]

[0030] The present invention also provides a composition comprising docosahexaenoic acid obtained by hydrolyzing DHA-containing oil obtained from fish or microalgae, and a method for decomposing and purifying oil obtained from fish or microalgae into polyunsaturated fatty acids.

[0031] In addition, the present invention provides a method for producing a dihydroxy fatty acid (resolvin D5) having 20 to 22 carbon atoms, including the steps of (1) hydrolyzing DHA-containing oil obtained from inexpensive fish or microalgae using a commercial lipase and an adsorption resin; (2) purifying the obtained hydrolyzate by a solvent extraction method to obtain a composition containing docosahexaenoic acid with high purity (about 70% to 99%); and (3) producing resolvin D5 through a whole-cell reaction with a transformed microorganism containing a double-15-lipooxidase gene using the purified composition as a substrate; and a method for purifying a dihydroxy fatty acid (resolvin D5) having 20 to 22 carbon atoms with high purity. The present invention also provides optimized conditions for producing resolvin D5 through a whole-cell reaction in the bioreactor.

[0032] The present invention includes a purification step (4) for producing resolvin D5 in a high yield after the step (3). According to the purification method, resolvin D5 can be obtained in a high yield of 90% or more (preferably 99% or more). The purification step (4) may be performed simultaneously or sequentially by ultrafiltration, column filtration, affinity chromatography, and reversed-phase chromatography. Preferably, ultrafiltration, reversed-phase chromatography, (preparative column filtration), and affinity chromatography may be performed in that order.

[0033] For the above affinity chromatography, an adsorption resin such as HP20, HP2MG, SP207, SP825, sp850 or XAD761 can be used, and for the above reversed phase chromatography, a C1(TMS) resin, a C4(Butyl) resin, a C8(Octyl) resin or a C18(Octadecyl) resin can be used, but is not limited thereto.

[0034] Specifically, the present invention provides a purification method for producing a hydrolyzate (a polyunsaturated fatty acid including docosahexaenoic acid) from the low-priced docosahexaenoic acid-enriched oil using a commercial lipase and an adsorption resin, and a composition including the same for obtaining docosahexaenoic acid with a purity of 90%.

[0035] The present invention provides a composition for producing a dihydroxy fatty acid having 20 to 22 carbon atoms (preferably, resolvin D5) by reacting the purified hydrolysate (polyunsaturated fatty acid including docosahexaenoic acid) as a substrate with a double 15-lipooxidase.

[0036] The present invention provides a composition for producing resolvin D5, a dihydroxy fatty acid, which contains high purity 95, 99% docosahexaenoic acid as a substrate and is an effective ingredient through a reaction with a double 15-lipooxidase enzyme.

[0037] In addition, the present invention provides a composition for producing resolvin D5, which comprises hydrolyzing oil obtained from fish or microalgae using a commercial lipase and an adsorption resin to obtain a polyunsaturated fatty acid including docosahexaenoic acid, and reacting the obtained 90% docosahexaenoic acid and high-purity 95% docosahexaenoic acid as a substrate with a double 15-lipid oxidation enzyme to produce resolvin D5, and then purifying the obtained resolvin D5 to a high purity using ultrafiltration, octadecyl-silica (ODS) resin, and prep-LC containing C18 resin.

[0038] In one embodiment of the present invention, the adsorption resin included in the composition may be an adsorption resin that reduces the substrate inhibition effect by binding to fatty acid during the reaction, and HP20, HP2MG, SP207, SP825, sp850, and XAD761 may be used, and SP207 is preferred.

[0039] In one embodiment of the present invention, it is preferable that the manufacturing method uses docosahexaenoic acid-fortified oil as a substrate at a concentration of 10 g / L.

[0040] In one embodiment of the present invention, the concentration of the adsorption resin SP207 in the above manufacturing method is preferably 10 g / L, but any adsorption resin may be used as the adsorption resin as long as it performs the role of an adsorption resin that binds to fatty acids, and specifically, SP207 may be used, but is not limited thereto.

[0041] In one embodiment of the present invention, the concentration of the lipase derived from Thermomyces lanuginosus, which is a commercial lipase, in the above manufacturing method is preferably 1000 U / mL, but any commercial lipase may be used as the lipase as long as it plays a role in hydrolyzing oil as a substrate into polyunsaturated fatty acid, and specifically, lipase derived from Thermomyces lanuginosus may be used, but is not limited thereto.

[0042] In addition, it is preferable that the above manufacturing method is carried out at a pH range of 6 to 9 (e.g., pH 8 to 8.5, preferably pH 8.5) and a temperature range of 10 to 40°C (preferably 20 or 30°C). To maintain these pH conditions, an EPPS buffer solution can be used as a reaction solvent.

[0043] In one embodiment, the ratio of hexane solvent and acetonitrile in the solvent extraction purification method is preferably 4:1 (v / v).

[0044] By maintaining these conditions, the productivity and purification yield of a composition containing a high purity polyunsaturated fatty acid (docosahexaenoic acid) from oil obtained from fish or microalgae can be improved.

[0045] The present invention provides a method for producing resolvin D5, a dihydroxy fatty acid, which comprises the step of treating a composition obtained by hydrolyzing and purifying oil obtained from fish or microalgae into a polyunsaturated fatty acid including docosahexaenoic acid through the whole-cell reaction in a flask and bioreactor with a recombinant strain expressing a double 15-lipolytic enzyme.

[0046] The present invention provides a method for producing resolvin D5, a dihydroxy fatty acid, which comprises a step of treating a recombinant strain expressing a double 15-lipooxidase with a high-purity 95, 99% docosahexaenoic acid reagent as a substrate in a flask and a bioreactor through the whole-cell reaction.

[0047] In the present invention, the dual 15-oxidation lipoprotein enzyme derived from an Archangium violaceum strain comprises an amino acid sequence of sequence number 1 or a sequence having at least 80% homology thereto, and sequence number 2 represents a gene sequence encoding the same.

[0048] In one embodiment, in the manufacturing method, the substrate may further include one selected from the group consisting of polyunsaturated fatty acids including eicosapentaenoic acid, docosapentaenoic acid, and arachidonic acid in addition to docosahexaenoic acid as a main component, as a docosahexaenoic acid-enriched oil, a docosahexaenoic acid reagent having a high purity of 95 or 99%, or a hydrolyzate hydrolyzed and purified from the docosahexaenoic acid-enriched oil.

[0049] In one embodiment, the composition may include a solvent and a polymer that effectively increases the solubility of fatty acids and substrates during the reaction, preferably dimethyl sulfoxide (DMSO) and polyvinyl pyrrolidone (PVP).

[0050] It is preferable that the above whole-cell reaction be carried out at a temperature in the range of 20°C. To maintain these pH conditions, an EPPS buffer solution can be used as a reaction solvent.

[0051] In addition, in the above manufacturing method, E. coli transformed with a recombinant expression vector containing a double 15-lipoproteinase gene may be cultured to induce expression of the recombinant enzyme gene, and then the strain containing the expressed protein may be recovered and used. It is preferable to obtain and use this. In addition, the time of the whole-cell reaction may be appropriately controlled according to a conventional method.

[0052] In another embodiment of the present invention, the manufacturing method is preferably used to treat cells having dual 15-lipolytic enzyme in a concentration range of 0.5 g / L to 6.0 g / L, but is not limited thereto.

[0053] The above manufacturing method is preferably, but not limited to, treating 90% pure docosahexaenoic acid as a hydrolyzate hydrolyzed and purified from the aforementioned high purity 95, 99% docosahexaenoic acid reagent or docosahexaenoic acid-enriched oil in the range of 1 mM to 9 mM.

[0054] The above manufacturing method preferably uses a concentration of dimethyl sulfoxide, which is a solvent, in the range of 0.1% to 10%, but any solvent may be used and is not limited thereto.

[0055] The above manufacturing method preferably processes the concentration of the polymer polyvinylpyrrolidone in the range of 0.1% to 10%, but any polymer may be used and is not limited thereto.

[0056] The above manufacturing method preferably treats the concentration of isopropyl-thiogalactopyranoside at a concentration of 0.1 mM, but is not limited thereto.

[0057] In addition, it is preferable that the above manufacturing method is carried out at a pH range of 6 to 9 (e.g., pH 8 to 8.5, preferably pH 8.5). To carry out the above reaction, an EPPS buffer solution can be used as a reaction solvent. Referring to FIG. 11A, it is suitable to carry out the reaction at a pH range of 6 to 9 (e.g., pH 8 to 8.5, preferably pH 8.5) depending on the reaction activity conditions of HEPPS and CHES.

[0058] The above whole-cell reaction is preferably performed at a temperature range of 10 to 40°C (approximately 20°C). Referring to FIG. 11B, when the reaction was performed for 30 minutes at various temperatures ranging from 15°C to 35°C in a 50 mM HEPPS (pH 8.5) buffer containing 0.5 g L-1 cells and 3 mM ARA, the reaction activity was higher when performed at 10 to 40°C (preferably 15 to 35°C, more preferably 15 to 25°C).

[0059] The above purification method is preferably, but not limited to, treating Resolvin D5 produced from a substrate with 90% purity docosahexaenoic acid as a hydrolyzate hydrolyzed and purified from a high-purity 95% docosahexaenoic acid reagent or docosahexaenoic acid-enriched oil in the range of 10 g / L.

[0060] In one embodiment, the purification method is preferably performed using ultrafiltration, C18 octadecyl, a production column, and adsorption resin SP825, but is not limited thereto.

[0061] In one embodiment, the concentration of the adsorption resin SP825 in the above purification method is preferably 10 g / L, but any adsorption resin may be used as the adsorption resin as long as it performs the role of an adsorption resin that binds to Resolvin D5, and specifically, SP825 may be used, but is not limited thereto.

[0062]

[0063] As described above, according to the present invention, docosahexaenoic acid of 90% purity was secured through hydrolyzed and purified hydrolyzate containing polyunsaturated fatty acids, including docosahexaenoic acid, using commercial lipase and adsorption resin from oil obtained from fish or microalgae, and a high-purity 95, 99% docosahexaenoic acid reagent as a substrate was used as a substrate, and the final product, resolvin D5 (7S, 17S-dihydroxy-4Z, 8E, 10Z, 13Z, 15E, 19Z-docosahexaenoic acid), was produced through an eco-friendly bioconversion method through a whole-cell reaction in which a dual 15-lipid oxidizing enzyme derived from Archangium violaceum strain was transformed. In the bioconversion process, the solubility of the substrate was efficiently increased by treating the solvent and polymer, and after producing it with high productivity and high yield through sufficient oxygen supply in the bioreactor, ODS and We confirmed that high-purity C18 resin can be manufactured. Consequently, by optimizing the reaction of resolvin D5, an endogenous substance in the human body, from oil obtained from inexpensive fish or microalgae, high yields of over 70% and purification yields of over 90% were produced in g / L units, and purification was possible at over 97%.

[0064] Resolvin D5, an endogenous human substance manufactured according to the present invention, acts as a signaling molecule and can participate in various physiological functions in animals, including humans. Using the manufacturing method of the present invention, resolvin D5 can be produced with higher productivity and yield than conventional methods, and purified to a high degree of purity, making it useful in various industrial fields, including pharmaceuticals, food, and cosmetics.

[0065] Figure 1 shows the results of the resolvin D5 production according to the concentration of dimethyl sulfoxide (DMSO), a solvent, and polyvinylpyrrolidone (PVP), a polymer, for maximum production of resolvin D5 produced by a whole-cell reaction transformed with a dual 15-lipooxidase derived from an Archangium violaceum strain using a high-purity 99% docosahexaenoic acid reagent as a substrate.

[0066] Figure 2 shows the results showing the production of resolvin D5 according to the concentration of whole cells and substrate for maximum production of resolvin D5 using a high-purity 99% docosahexaenoic acid reagent as a substrate.

[0067] Figure 3 shows the results of the production of resolvin D5 according to the types of solvents and polymers for maximum production of resolvin D5 when polyunsaturated fatty acids containing docosahexaenoic acid with a purity of 90% were used as substrates for bioconversion after hydrolysis and solvent extraction of oil obtained from fish or microalgae. Isopropyl alcohol (IPA), methanol (MeOH), ethanol (ETOH), ethyl acetate (EA), and dimethyl sulfoxide (DMSO) were used as solvents, and polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), and polyethylene glycol (PEG) were used as polymers.

[0068] Figure 4 shows the results of the resolvin D5 production according to the concentration of the solvent dimethyl sulfoxide and the polymer polyvinylpyrrolidone for maximum production of resolvin D5 produced by a whole-cell reaction transformed with a dual 15-lipooxidase derived from an Archangium violaceum strain using polyunsaturated fatty acids containing docosahexaenoic acid with a purity of 90%, obtained by hydrolyzing and solvent-extracting oil obtained from fish or microalgae.

[0069] Figure 5 shows the results of resolvin D5 production according to the concentration of whole cells and substrate for maximum production of resolvin D5 using docosahexaenoic acid with a purity of 90% in the hydrolyzate as a substrate.

[0070] Figure 6 shows the results of the production of resolvin D5 according to the concentration of whole cells and substrate for maximum production of resolvin D5 using a high-purity 95% docosahexaenoic acid reagent as a substrate in a whole-cell reaction in a bioreactor for mass production.

[0071] Figure 7 shows the results of the production of resolvin D5 over time by recombinant E. coli expressing dual 15-lipooxidase derived from Archangium violaceum strain using a high-purity 95% docosahexaenoic acid reagent as a substrate in a bioreactor for mass production.

[0072] Figure 8 shows the results of the resolvin D5 production according to the concentration of whole cells and substrate for maximum production of resolvin D5 produced using docosahexaenoic acid with a purity of 90% as a substrate in the hydrolysate of oil obtained from fish or microalgae through whole-cell reaction in a bioreactor for mass production.

[0073] Figure 9 shows the production of resolvin D5 over time by recombinant E. coli expressing dual 15-lipooxidase derived from Archangium violaceum strain using docosahexaenoic acid with a purity of 90% as a substrate in a hydrolyzate of oil obtained from fish or microalgae in a bioreactor.

[0074] Figure 10 shows a recombinant expression vector that synthesizes a dual 15-lipooxidase derived from an Archangium violaceum strain according to the present invention.

[0075] Figure 11 shows the results of examining the effects of pH, temperature, and cysteine ​​concentration on 5S,15S-DiHETE production. Figure 11A is a graph examining the effect of pH on 5S,15S-DiHETE (5S,15S-dihydroxyeicosatetraenoic acid) production. The reaction was performed at 20°C for 30 minutes in 50 mM 3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid (HEPPS) buffer and 50 mM N-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer, while changing the pH from 7.5 to 9.5, containing 0.5 g L-1 cells and 3 mM ARA. In Figure 11A, symbols represent 50 mM HEPPS (●) and 50 mM CHES (○). Figure 11B shows the results of examining the effect of temperature on 5S,15S-DiHETE production. In this example, the reaction was performed at various temperatures from 15°C to 35°C for 30 minutes in a 50 mM HEPPS (pH 8.5) buffer containing 0.5 g L-1 cells and 3 mM ARA.

[0076] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0077]

[0078] [Example 1]

[0079] Preparation of double 15-lipolytic enzyme

[0080] The transformed microorganisms were cultured in 1 L of Luria-Bertani (LB; Difco, Sparks, MD, USA) medium containing 20 μg / ml kanamycin in a shaker flask at 37°C under aeration conditions of 200 rev / min. When the whole-cell absorbance reached 0.6 to 0.8 at 600 nm, 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce protein expression of the enzymes, and the culture was cultured for 16 h at 16°C with shaking at 150 rev / min. In addition, the dual 15-oxidized lipoprotein enzyme produced by overexpression as described above was centrifuged at 6,000× gravitational acceleration (g) at 4°C for 30 minutes in a culture medium of the transformed strain, washed twice with 0.85% sodium chloride (NaCl), and then used as a recombinant cell for producing resolvin D5.

[0081]

[0082] [Example 2]

[0083] Production of resolvin D5 from 99% pure docosahexaenoic acid reagent using double 15-liposide oxidase.

[0084] To mass-produce resolvin using the above-mentioned dual 15-oxidation lipoprotein enzyme, the reaction was performed using docosahexaenoic acid as a substrate. To maximize the production of resolvin D5 produced by the whole-cell reaction transformed with the dual 15-oxidation lipoprotein enzyme derived from the Archangium violaceum strain, the optimal concentrations of the solvent dimethyl sulfoxide and the polymer polyvinylpyrrolidone were identified. The whole-cell reaction was performed at pH 8.5, 20℃ for 60 minutes, including 3 mM docosahexaenoic acid and 1 g / L whole cells.

[0085] As a result, it was confirmed that the optimal concentrations of the solvent dimethyl sulfoxide and the polymer polyvinylpyrrolidone in Fig. 1 were each 1%. In addition, the maximum production was confirmed through optimization of the concentration of the recombinant E. coli expressing the double 15-oxidation lipoprotein enzyme and the concentration of the substrate docosahexaenoic acid reagent with 99% purity under the optimal concentrations of the solvent and polymer. As a result, it was confirmed that the optimal concentrations of the recombinant E. coli expressing the double 15-oxidation lipoprotein enzyme and the substrate docosahexaenoic acid reagent with 99% purity were 1 g / L and 3 mM, respectively, in Fig. 2. It was confirmed that approximately 2.24 mM (0.81 g / L) of resolvin D5 was produced in the flask by the whole-cell reaction in which the double 15-oxidation lipoprotein enzyme was expressed (Fig. 2).

[0086]

[0087] [Example 3]

[0088] Hydrolysis and purification of polyunsaturated fatty acids from docosahexaenoic acid-enriched oil

[0089] 10 g / L of oil obtained from inexpensive fish or microalgae was hydrolyzed at 30°C for 24 hours with stirring at 250 rpm using 1000 U / ml of commercial lipase and 10 g / L of adsorption resin SP207 to decompose into polyunsaturated fatty acids containing docosahexaenoic acid with a purity of 70%. To increase the purity, the hydrolyzate was added with a 4:1 (v / v) ratio of hexane solvent and acetonitrile, stirred for 4 minutes, stored at -20°C for 2 hours, and then the lower acetonitrile layer was obtained to secure a polyunsaturated fatty acid substrate containing docosahexaenoic acid with a purity of 90% (Table 1).

[0090] Table 1 below shows the results of purification through hydrolysis and solvent extraction of polyunsaturated fatty acids containing 90% purity docosahexaenoic acid from oil obtained from fish or microalgae using commercial lipase and adsorption resin.

[0091] [Correction pursuant to Rule 91, October 16, 2024]

[0092]

[0093] [Example 4]

[0094] Synthesis of resolvin D5 using polyunsaturated fatty acids containing 90% pure docosahexaenoic acid, hydrolyzed and solvent-extracted from docosahexaenoic acid-enriched oil, as a substrate.

[0095] The optimal production amount for each type of solvent and polymer was confirmed for maximum production of resolvin D5 using polyunsaturated fatty acids containing 90% pure docosahexaenoic acid, which was hydrolyzed and solvent-extracted from docosahexaenoic acid-enriched oil using the above-mentioned dual 15-oxidation lipoprotein enzyme. In order to confirm the optimal type of solvent and polymer suitable for production, 1.0 g / L of whole cells transformed with recombinant dual 15-oxidation lipoprotein enzyme was used using 2 mM 90% pure docosahexaenoic acid as a substrate, and whole cell reaction was performed at pH 8.5, 20℃ for 60 minutes. As a result, it was confirmed in Fig. 3 that dimethyl sulfoxide as a solvent and polyvinylpyrrolidone as a polymer were the optimal types of additives for the production of resolvin D5. A whole cell reaction was performed at pH 8.5 and 20°C for 60 min using 90% pure docosahexaenoic acid, which was solvent-extracted after 2.0 mM hydrolysis, as a substrate and 1 g / L of recombinant dual 15-lipolipase-transformed whole cells. The optimal concentrations of the additives were confirmed to be 1.0% dimethyl sulfoxide and 5% polyvinylpyrrolidone, respectively (Fig. 4).

[0096] In addition, the maximum production was confirmed through concentration optimization of the recombinant E. coli expressing dual 15-oxidation lipoprotein enzyme at the optimal concentration of solvent and polymer and the polyunsaturated fatty acid containing docosahexaenoic acid with a purity of 90%, which was hydrolyzed and solvent-extracted from oil obtained from fish or microalgae, as a substrate. As a result, in Fig. 5, the optimal concentration of the recombinant E. coli expressing dual 15-oxidation lipoprotein enzyme and the optimal concentration of docosahexaenoic acid with a purity of 90%, which was hydrolyzed and solvent-extracted as a substrate were confirmed to be 1 g / L and 2 mM, respectively. It was confirmed that about 1.8 mM (0.65 g / L) of resolvin D5 was produced in the flask by the whole-cell reaction in which dual 15-oxidation lipoprotein enzyme was expressed (Fig. 5).

[0097]

[0098] [Example 5]

[0099] Mass production of resolvin D5 using a whole-cell reaction of dual 15-oxidation lipase using polyunsaturated fatty acids containing 95% pure docosahexaenoic acid reagent and 90% pure docosahexaenoic acid solvent-extracted from oil obtained from fish or microalgae as substrates in a bioreactor.

[0100] (lipoxygenase, LOX) containing 90% pure docosahexaenoic acid, hydrolyzed from 95% pure docosahexaenoic acid reagent and docosahexaenoic acid-enriched oil by recombinant Escherichia coli expressing dual 15-lipolytic enzyme in 1 L culture medium in a 3 L bioreactor. Whole-cell reactions were performed for 6 hours at pH 8.5, 20°C, 1000 rpm, and 1.0 vvm using 95% pure docosahexaenoic acid as a substrate under optimal conditions of 1.0% dimethyl sulfoxide and 1.0% polyvinyl pyrrolidone. In addition, 90% pure docosahexaenoic acid, which was hydrolyzed from oil and then solvent-extracted, was used as a substrate under 1.0% dimethyl sulfoxide and 5.0% polyvinyl pyrrolidone, and the production of resolvin D5 was confirmed through optimization of whole-cell concentration and substrate concentration in a 3 L bioreactor for each reaction.

[0101] As a result, as shown in Fig. 6, it was confirmed that the highest concentration of resolvin D5 was produced when 1.6 g / L of whole cells were treated with 5.0 mM high-purity 95% docosahexaenoic acid reagent as a substrate, and among these, it was confirmed that a maximum of approximately 4.21 mM (1.52 g / L) of resolvin D5 was produced by the whole-cell reaction in which 15-oxidation lipase was expressed (Fig. 7). In addition, as shown in Fig. 8, it was confirmed that the highest concentration of resolvin D5 was produced when 2.5 g / L of whole cells were treated with 5.0 mM high-purity 95% docosahexaenoic acid as a substrate, and among these, it was confirmed that a maximum of approximately 3.97 mM (1.43 g / L) of resolvin D5 was produced by the whole-cell reaction in which 15-oxidation lipase was expressed (Fig. 9). This was confirmed to be approximately 197% and 217% higher than the production in the flask using a 3L bioreactor that facilitates oxygen addition.

[0102]

[0103] [Example 6]

[0104] Purification and yield verification of mass-produced resolvin D5 using ultrafiltration, C18 octadecyl, manufacturing columns, and adsorption resins.

[0105] To increase the purity of resolvin D5 in the 1 L reaction solution mass-produced using the above bioreactor, a four-step purification process was performed using ultrafiltration, C18 octadecyl, a preparative column, and adsorption resin SP825 (Table 2). First, to remove impurities and high-molecular-weight proteins, ultrafiltration was performed using a 10 kDa filter, followed by centrifugation at 6,000 × g for 30 min at 4°C. The filtered material was stabilized with triple-distilled water on the C18 octadecyl resin, and the primary purified product was washed with a 50% acetonitrile solvent, and then resolvin D5 was purified with a 60% acetonitrile solvent to obtain it. Resolvin D5, which had undergone secondary purification using C18 octadecyl resin, was purified using a preparative column with a concentration gradient of 50% and 100% acetonitrile solvent at a flow rate of 6 mL / min at 30°C for 30 min, and purified at an absorbance value of 202 nm. Finally, the previously purified Resolvin D5 was mixed with 10 g / L adsorption resin SP825 at 40°C under stirring conditions of 200 rpm for 3 h, and the resin was recovered through a 45 μm filter, and the adsorption resin with Resolvin D5 attached was dissolved in ethyl acetate to obtain the product. Resolvin D5 produced from 90% pure docosahexaenoic acid, which was hydrolyzed and solvent-extracted through four purification processes by the above method, had a purification yield improved by 25.3% compared to the control group, and resolvin D5 produced from 95% pure docosahexaenoic acid reagent had a purification yield improved by 23.1% compared to the control group (Table 2).

[0106] Table 2 below shows the results of the purity, yield, and recovery rate at each step during the purification process using ultrafiltration, C18 octadecyl, production column, and adsorption resin of resolvin D5 produced in a bioreactor.

[0107]

[0108] As described above, the present invention relates to a method for producing resolvin D5, an endogenous substance in the human body, in a bioconversion process, by hydrolyzing polyunsaturated fatty acids including docosahexaenoic acid using commercial lipase and an adsorption resin from oil obtained from fish or microalgae, and then obtaining a docosahexaenoic acid substrate with a purity of 90% through a solvent extraction method. Including this as a substrate, the present invention also relates to a method for producing resolvin D5 in units of g / L using a bioconversion process, by optimizing the reaction conditions for conversion into resolvin D5, an endogenous substance in the human body, in a flask and bioreactor using a dual arachidonic acid 15-oxidation lipase derived from the bacterium Archangium violaceum, using 95 and 99% docosahexaenoic acid reagents as substrates, respectively, through a whole-cell reaction. Specifically, the substrate is a hydrolyzate containing docosahexaenoic acid-enriched oil, a docosahexaenoic acid reagent with a high purity of 95, 99%, and a docosahexaenoic acid with a purity of 90%, and the transformed cells containing a dual arachidonic acid 15-oxidation lipase from Archangium violaceum are used to produce an improved concentration of resolvin D5 in a reaction mixture with the addition of a solvent, dimethyl sulfoxide, and a polymer, polyvinylpyrrolidone, using a bioconversion method in a flask and a bioreactor. Therefore, compared to conventional chemical methods, it is not only environmentally friendly, but also has great significance in that it is a specific mass production of an endogenous human substance that has not been previously reported by a bioconversion method.

[0109]

[0110] While specific aspects of the present invention have been described in detail, it will be apparent to those skilled in the fatty acid and pharmaceutical industries that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0111]

[0112] Sequence number 1

[0113] 15-lipoxygenase derived from Archangium violaceum strain

[0114] MRSIPSLPQN DSPAQQELRL ATLTQERQVY AYNFDSRLSP LGIAAQVPRQ DNFSFVWLDG

[0115] VATTGLQLLG NVLAIGAKLF DNGEAIQNDA GLSELELRRV EGTYRELTDG FSRLSDRTSR

[0116] LHQAPRSPAV TLIADVIERP RTLLKAAGQK LEETAGVAIV SELATQAALD APAIGQDILD

[0117] LIDTLLKKLL SEAGDLFLKY LGLYGKAASL DNYTAQFSLL QPPSVASNYE TDLIFARMRL

[0118] AGPNPALLQG IAALPEKFPV TDAQYQSVMG SQDTLARAGQ EGRLYLLDYA VFQGIPTGQT

[0119] SGGQKYIEAP LALFAVPAAN QADRKLRAVA IQCSQTPGRS NPIFTPADGT SWSLARLHVQ

[0120] VADGNYHELI AHLGLTHLIL EEFTLSTHRQ LAPQHPLYLL LTPHFQGTLA INNAAETSLI

[0121] <h2 style=";text-align:left;direction:ltr">APGGPVDQLL AGEIKASTQV SIQAVANQSI NQSFLPRALA ARGVDDASKL PDYPYRDDAL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0122] <h2 style=";text-align:left;direction:ltr"> LLWNDIRAWV SDYLAIYYND DAAVRSDYEL QAWVTELASP DAGKLKDVTE GGGGIQTFEY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0123] <h2 style=";text-align:left;direction:ltr"> LVDLTTYIIF TASVQHAAVN FPQRTVMSYT PALPLAAYAP APTSVEELPP STELTHLPPL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0124] <h2 style=";text-align:left;direction:ltr"> QMAFLQQAVT FGLGNVYFTR LGGYDTYLRE PWFADARVWP ALEVFQKRLR ATEREIGQRN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0125] <h2 style=";text-align:left;direction:ltr"> LSRIPYETLL PTAIPQSINI Z<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0126] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0127] <h2 style=";text-align:left;direction:ltr"> 서열번호 2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0128] <h2 style=";text-align:left;direction:ltr"> 15-lipoxygenase derived from Archangium violaceum strain<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0129] <h2 style=";text-align:left;direction:ltr"> atgcgttcca ttccctccct gccccagaat gactctcccg cccagcagga gctgcgcctc<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0130] <h2 style=";text-align:left;direction:ltr"> gccacgctca cacaagagcg gcaggtctac gcctacaact tcgacagccg gctgagtccg<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0131] <h2 style=";text-align:left;direction:ltr"> ctggggatcg cggcccaggt cccccgtcag gacaacttct ccttcgtctg gttggatggc<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0132] <h2 style=";text-align:left;direction:ltr"> gtcgccacca ccggcctgca gctgctgggc aacgtgctgg ccatcggcgc gaagctcttc<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0133] <h2 style=";text-align:left;direction:ltr"> gacaacgggg aggcgatcca gaacgacgcc ggcctgtccg agctggagct ccgcagggtc<h2 style=";text-align:left;direction:ltr">

[0134] gaggggacct accgggagct gaccgacgga ttcagccggc tgtcggaccg gacctcccgg

[0135] ctccaccagg cgccgcgctc cccggcggtg acgctcatcg ccgatgtcat cgagcgcccc

[0136] cggacgctgc tcaaggcggc gggccagaag ctggaggaga ccgccggtgt cgccatcgtc

[0137] tccgagctcg ccacccaggc cgcgctggat gcgcccgcca tcgggcagga catcctcgac

[0138] ctcatcgaca ccctgttgaa gaagctgttg tccgaggccg gagatctgtt cctgaagtac

[0139] ctggggctgt acggcaaggc ggcgtcgctc gacaactaca cggcccagtt ctcgctgctg

[0140] cagcctccgt cggtcgccag caattacgag accgacctca tcttcgcgcg catgcggctg

[0141] gccgggccca acccggcgct gctccagggc atcgcggccc tccccgagaa gttcccggtg

[0142] acggacgcgc agtaccagtc cgtgatgggc tcgcaggaca ccctggcccg cgcgggccag

[0143] gaaggtcggc tgtacctgct ggactacgcc gtcttccagg gcatccccac cggacagacg

[0144] tcgggcggac agaagtacat cgaagcgccg ctcgccctgt tcgccgtgcc cgccgcgaac

[0145] caggccgacc ggaagctgcg cgcggtggcc atccagtgct cgcagacgcc gggccgctcc

[0146] aaccccatct tcaccccggc ggacggcacc tcctggagcc tggcgcgcct gcacgtgcag

[0147] gtggcggatg gcaactacca cgagctcatc gcgcacctgg gcctcacgca cctgattctg

[0148] gaagagttca ccctgtccac gcaccggcag ctcgcgcccc agcacccgct ctacctgctg

[0149] ctgacgccgc acttccaggg caccctggcc atcaacaacg ccgcggagac gtccctcatc

[0150] gcgcccggtg gcccggtgga ccagctgctc gccggtgaga tcaaggcctc cacccaggtg

[0151] agcatccagg ccgtggccaa ccaatccatc aaccagtcgt tcctgccccg ggcgctggcg

[0152] gcccgcgggg tggacgacgc ctcgaagctg ccggactacc cgtaccgcga cgacgcgctg

[0153] ctgctctgga acgacatccg cgcctgggtg agcgactacc tggccatcta ctacaacgat

[0154] gacgcggcgg tgcggagcga ctacgagctg caggcctggg tcacggagct cgccagcccc

[0155] gacgccggca agctgaagga tgtgacggag ggcggagggg gcatccagac cttcgagtat

[0156] ctggtggacc tgaccaccta catcatcttc acggcgagcg tgcagcacgc ggcggtcaac

[0157] ttcccgcagc gcacggtcat gagctacacc ccggcgctgc cgctggcggc ctacgctccc

[0158] gcgcccacct ccgtggaaga gctgccgccg agcacggagc tgacccacct gccgccgttg

[0159] cagatggcct tcctgcagca ggcggtcacc ttcgggctgg gcaacgtgta cttcacccgg

[0160] ctgggcggct acgacacgta cctgcgcgag ccctggttcg cggacgcgcg cgtgtggccc

[0161] gccctggagg tcttccagaa gcgcctgcgc gccacggagc gggagatcgg ccagcgcaac

[0162] ctgtcgcgca tcccctacga gacgctgctg cccacggcga ttccgcagag catcaacatc

[0163] taa

Claims

1. A composition for producing a dihydroxy fatty acid having 20 to 22 carbon atoms, comprising a polyunsaturated fatty acid derived from oil containing DHA obtained from fish or microalgae with high purity.

2. In paragraph 1, A composition wherein the polyunsaturated fatty acid is obtained by hydrolyzing oil containing DHA obtained from the fish or microalgae.

3. In paragraph 2, A composition wherein the hydrolyzate of the DHA-containing oil obtained from the above fish or microalgae is obtained by hydrolyzing the DHA-containing oil with lipase and an adsorption resin.

4. In paragraph 3, A composition wherein the lipase is a lipase derived from Thermomyces lanuginosus, and the adsorption resin is SP207, HP20, HP2MG, SP825, sp850, or XAD761.

5. In paragraph 2, A composition wherein the polyunsaturated fatty acid is obtained by purifying a hydrolyzate of DHA-containing oil obtained from the fish or microalgae using a solvent in which hexane and acetonitrile are mixed in a volume ratio of 3 to 5:

1.

6. In paragraph 1, A composition comprising at least one fatty acid selected from the group consisting of docosahexaenoic acid, eicosapentaenoic acid, docosapentaenoic acid, and arachidonic acid.

7. In paragraph 1, A composition comprising a dihydroxy fatty acid having 20 to 22 carbon atoms and resolvin D5.

8. In paragraph 1, The composition above is a composition containing docosahexaenoic acid as a polyunsaturated fatty acid with a high purity of 70 to 99%.

9. A method for producing resolvin D5, a) A step of producing a vector encoding a dual 15-oxidation lipase gene derived from an Archangium violaceum strain; b) a step of transforming a microorganism with a vector produced according to step a); c) A method comprising a step of producing resolvin D5 through bioconversion by reacting a composition containing a high purity polyunsaturated fatty acid derived from DHA-containing oil obtained from fish or microalgae with a microorganism transformed according to step b) above.

10. In paragraph 9, A method comprising, after step c), a purification step d) for producing resolvin D5 in high yield.

11. In paragraph 10, The above d) purification step is a method in which purification is performed simultaneously or sequentially by ultrafiltration, column filtration, affinity chromatography, and reverse phase chromatography.

12. In paragraph 10, The above affinity chromatography is a method for purifying using an adsorption resin of HP20, HP2MG, SP207, SP825, sp850 or XAD761.

13. In paragraph 10, The above reverse phase chromatography is a method for purifying using C1(TMS) resin, C4(Butyl) resin, C8(Octyl) resin or C18(Octadecyl) resin.

14. In paragraph 9, A method wherein the polyunsaturated fatty acid comprises at least one fatty acid selected from the group consisting of docosahexaenoic acid, eicosapentaenoic acid, docosapentaenoic acid, and arachidonic acid.

15. In paragraph 9, The composition of the above step c) is a method in which the composition contains docosahexaenoic acid as a polyunsaturated fatty acid with a high purity of 70 to 99%, and is obtained by hydrolyzing DHA-containing oil with lipase and an adsorption resin.

16. In paragraph 9, A method wherein the double 15-lipolytic enzyme of step a) above is composed of an amino acid sequence of sequence number 1 or a sequence having 80% homology with the amino acid sequence of sequence number 1.

17. In paragraph 15, A method in which the above polyunsaturated fatty acid is obtained by purifying a hydrolyzate of DHA-containing oil obtained from the above fish or microalgae with a solvent in which hexane and acetonitrile are mixed in a volume ratio of 3 to 5:

1.

18. In paragraph 15, A method in which lipase is treated at a concentration of 800 to 1200 U / ml when the concentration of DHA-containing oil obtained from fish or microalgae during the above hydrolysis is 5 to 15 g / L.

19. In paragraph 9, The above method is a method in which the reaction is performed in the range of pH 6 to 9.

20. In paragraph 9, The above method is a method in which the reaction is performed at a temperature range of 10 to 40°C.

21. In paragraph 9, A method in which a solvent and a polymer are treated to increase the solubility of the substrate during the bioconversion of step c).

22. In paragraph 21, A method wherein the solvent comprises at least one selected from the group consisting of isopropyl alcohol (IPA), methanol (MeOH), ethanol (EtOH), ethyl acetate (EA), and dimethyl sulfoxide (DMSO).

23. In paragraph 22, A method wherein the concentration of dimethyl sulfoxide (DMSO) contained in the above solvent is 0.1% to 10%.

24. In paragraph 21, A method wherein the polymer comprises at least one selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and polyethylene glycol (PEG).

25. In paragraph 24, A method wherein the polymer is polyvinylpyrrolidone (PVP), and the concentration of polyvinylpyrrolidone to be treated is 0.1% to 10%.

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