KR102555535B1 - Composition and method for producing hydroxy fatty acid or dihydroxy fatty acid using 9-lipoxygenase or variant thereof - Google Patents

Composition and method for producing hydroxy fatty acid or dihydroxy fatty acid using 9-lipoxygenase or variant thereof Download PDF

Info

Publication number
KR102555535B1
KR102555535B1 KR1020220062926A KR20220062926A KR102555535B1 KR 102555535 B1 KR102555535 B1 KR 102555535B1 KR 1020220062926 A KR1020220062926 A KR 1020220062926A KR 20220062926 A KR20220062926 A KR 20220062926A KR 102555535 B1 KR102555535 B1 KR 102555535B1
Authority
KR
South Korea
Prior art keywords
acid
lipoxygenase
ala
fatty acid
leu
Prior art date
Application number
KR1020220062926A
Other languages
Korean (ko)
Other versions
KR20220075286A (en
Inventor
오덕근
김성은
Original Assignee
건국대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 건국대학교 산학협력단 filed Critical 건국대학교 산학협력단
Priority to KR1020220062926A priority Critical patent/KR102555535B1/en
Publication of KR20220075286A publication Critical patent/KR20220075286A/en
Application granted granted Critical
Publication of KR102555535B1 publication Critical patent/KR102555535B1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C57/00—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms
    • C07C57/02—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms with only carbon-to-carbon double bonds as unsaturation
    • C07C57/03—Monocarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
    • C12Y113/11—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of two atoms of oxygen (1.13.11)
    • C12Y113/11058—Linoleate 9S-lipoxygenase (1.13.11.58)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)

Abstract

본 발명은 신규 미생물 유래 9-리폭시게나아제를 이용하여 생물전환 공정으로 신규 수산화지방산 또는 이수산화지방산을 제조하기 위한 것으로, 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) 유래 9-리폭시게나아제; 또는 상기 9-리폭시게나아제의 이마노산 서열로부터, 569번째 아미노산인 발린(V)을 페닐알라닌(F)로 치환시킨 9-리폭시게나아제 변이체를 유효성분으로 포함하는 수산화지방산 또는 이수산화지방산 제조용 조성물 및 제조방법에 관한 것이다. The present invention is to prepare a novel hydroxylated fatty acid or dihydrogenated fatty acid by a bioconversion process using 9-lipoxygenase derived from a novel microorganism, Sphingomonas macrogoltabidus 9-lipoxygenase derived from Sphingomonas macrogoltabidus; Or a composition for producing hydroxylated fatty acid or dihydroxidized fatty acid comprising, as an active ingredient, a 9-lipoxygenase variant in which valine (V), the 569th amino acid, is substituted with phenylalanine (F) from the imanoic acid sequence of the 9-lipoxygenase, and It's about manufacturing methods.

Description

9-리폭시게나아제 또는 이의 변이체를 이용한 수산화지방산 또는 이수산화지방산 제조용 조성물 및 이를 이용한 제조방법{COMPOSITION AND METHOD FOR PRODUCING HYDROXY FATTY ACID OR DIHYDROXY FATTY ACID USING 9-LIPOXYGENASE OR VARIANT THEREOF}Composition for producing hydroxylated fatty acid or dihydroxylated fatty acid using 9-lipoxygenase or a variant thereof and method for producing hydroxylated fatty acid using the same

본 발명은 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) 유래 9-리폭시게나아제; 또는 상기 9-리폭시게나아제가 위치 지정 돌연변이에 의해 변형된 9-리폭시게나아제 변이체(V569F)를 이용한 수산화지방산 또는 이수산화지방산 제조용 조성물 및 제조방법에 관한 것이다.The present invention is Sphingomonas macrogoltabidus ( Sphingomonas macrogoltabidus ) Derived 9-lipoxygenase; Alternatively, it relates to a composition and method for preparing hydroxyl fatty acid or dihydroxyl fatty acid using a 9-lipoxygenase variant (V569F) in which the 9-lipoxygenase is modified by site-directed mutation.

수산화지방산은 동물, 식물, 곤충 그리고 미생물 등 자연계의 여러 생물체 지질에 존재하는 물질로 일반적으로 지방산에 수산화기를 가지고 있는 물질이다. 수산화 지방산은 수산화기에 의해 반응성이 뛰어나 산업적으로 원료물질로 사용되며, 수산화기의 작용으로 표면장력의 감소, 항균 및 항진균력 활성이 높아 화장품의 원료물질로 사용된다. 여러 생물체 중 특히 인간에게 발견되는 수산화 지방산은 인체 내의 신호전달물질의 전구체로 이용되며, 단일 물질만으로도 다양한 생리활성에 관여한다. 인간 신호전달물질의 한 종류인 지질조절제(lipid mediator)는 인간을 포함한 포유류 내에서 항상성 조절, 면역반응 등 다양한 생리활성 기능에 관여하는 중요한 물질이다.Hydroxylated fatty acids are substances present in lipids of various organisms in the natural world, such as animals, plants, insects, and microorganisms, and generally have a hydroxyl group in a fatty acid. Hydroxylated fatty acids are industrially used as raw materials due to their high reactivity by hydroxyl groups, and are used as raw materials for cosmetics due to the action of hydroxyl groups, which reduce surface tension and have high antibacterial and antifungal activity. Hydroxylated fatty acids found in humans, among many organisms, are used as precursors for signaling substances in the human body, and are involved in various physiological activities with just a single substance. Lipid mediators, a type of human signaling substance, are important substances involved in various physiological functions such as homeostasis regulation and immune response in mammals including humans.

리폭시게나아제(lipoxygenase, LOX)는 이산소화효소(dioxygenase)로 산화효소임에도 헴(heme)을 지니지 않고 철을 함유하는 것이 특징으로, 불포화지방산을 산소가 두 개 있는 과산화지방산(hydroperoxy fatty acid)으로 전환시키고 전환된 과산화지방산은 자연상태에서 산소가 한 개 있는 수산화지방산으로 촉매하는 다기능 효소이다. 특징적으로 다수의 cis, cis-1,4-펜타디엔을 가지는 다가불포화지방산만을 기질로 이용하여 이산소화 반응을 통해 입체특이성과 반응특이성을 촉매한다. 기질로 사용되는 다가불포화지방산의 종류에 따라 위치특이성이 다르며, 주로 탄소수 20개의 불포화지방산인 아리키돈산에 산화시키는 위치에 따라 위치특이적 산화효소로 명명되며 지금까지 밝혀진 리폭시게나아제는 위치특이성에 따라 5-, 8-, 11-, 12-, 15-리폭시게나아제가 존재한다. 그 중에서도 동물성 다가불포화지방산의 9번 위치에 수산기를 생산하는 9-리폭시게나아제(9-lipoxygenase)의 경우, 특이적으로 아라키돈산과 같은 탄소수 20개 이상의 불포화지방산에서 9번 탄소 위치에 수산기를 형성한다. 또한 이 효소는 탄소수 22개 이상의 불포화 지방산에서 11번 탄소 위치에 수산기를 형성한다. 9-리폭시게나아제의 경우 현재까지 인간을 포함한 동물에서는 발견된 바가 없고 홍조류(red algae) 등 해양생물에서 9-수산화아라키돈산이 보고되었지만 9-리폭시게나아제에 대한 동정 및 생화학적 특성은 보고된 바가 없다. Lipoxygenase (LOX) is a dioxygenase, and although it is an oxidase, it is characterized by containing iron without heme. Conversion and converted fatty acids are multifunctional enzymes that naturally catalyze hydroxyl fatty acids with one oxygen. Characteristically, only polyunsaturated fatty acids having a large number of cis, cis-1,4-pentadiene are used as substrates, and stereospecificity and reaction specificity are catalyzed through dioxygenation. Regiospecificity differs depending on the type of polyunsaturated fatty acid used as a substrate. It is mainly named as regiospecific oxidase depending on the position of oxidizing arichidonic acid, an unsaturated fatty acid with 20 carbon atoms. 5-, 8-, 11-, 12-, 15-lipoxygenases exist. Among them, 9-lipoxygenase, which produces a hydroxyl group at position 9 of animal polyunsaturated fatty acids, specifically forms a hydroxyl group at position 9 in unsaturated fatty acids having 20 or more carbon atoms, such as arachidonic acid. . In addition, this enzyme forms a hydroxyl group at the 11th position in unsaturated fatty acids with 22 or more carbon atoms. In the case of 9-lipoxygenase, it has not been found in animals including humans, and 9-hydroxyarachidonic acid has been reported in marine organisms such as red algae, but identification and biochemical characteristics of 9-lipoxygenase have been reported. does not exist.

본 발명은 신규 미생물 유래 9-리폭시게나아제를 이용하여 생물전환 공정으로 신규 수산화지방산 또는 이수산화지방산을 제조하기 위한 것으로, 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) 유래 9-리폭시게나아제; 또는 상기 9-리폭시게나아제의 이마노산 서열로부터, 569번째 아미노산인 발린(V)을 페닐알라닌(F)로 치환시킨 9-리폭시게나아제 변이체를 유효성분으로 포함하는 수산화지방산 또는 이수산화지방산 제조용 조성물 등을 제공하는 것이다. The present invention is to prepare a novel hydroxylated fatty acid or dihydrogenated fatty acid by a bioconversion process using 9-lipoxygenase derived from a novel microorganism, Sphingomonas macrogoltabidus 9-lipoxygenase derived from Sphingomonas macrogoltabidus; Or a composition for preparing hydroxylated fatty acid or dihydroxidized fatty acid comprising, as an active ingredient, a 9-lipoxygenase variant in which valine (V), the 569th amino acid, is substituted with phenylalanine (F) from the imanoic acid sequence of the 9-lipoxygenase. is to provide

그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

본 발명은 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) 유래 9-리폭시게나아제; 또는 상기 9-리폭시게나아제의 이마노산 서열로부터, 569번째 아미노산인 발린(V)을 페닐알라닌(F)로 치환시킨 9-리폭시게나아제 변이체를 유효성분으로 포함하는 수산화지방산 또는 이수산화지방산 제조용 조성물로서, 상기 수산화지방산 또는 이수산화지방산은 9-수산화아라키돈산(9-hydroxyarachidonic acid), 9,15-이수산화아라키돈산(9,15-dihydroxyarachidonic acid), 9-수산화에이코사펜타엔산(9-hydroxyeicopentaenoic acid), 9,15-이수산화에이코사펜타엔산(9,15-dihydroxyeicopentaenoic acid), 11-수산화도코사펜타엔산(11-hydroxydocosapentaenoic acid), 11,17-이수산화도코사펜타엔산(11,17-dihydroxydocosapentaenoic acid), 11-수산화도코사헥사엔산(11-hydroxydocosahexaenoic acid) 및 11,17-이수산화도코사헥사엔산(11,17-dihydroxydocosahexaenoic acid)으로 이루어진 군으로부터 선택된 하나 이상인, 수산화지방산 또는 이수산화지방산 제조용 조성물을 제공한다. The present invention is Sphingomonas macrogoltabidus ( Sphingomonas macrogoltabidus ) Derived 9-lipoxygenase; Or a hydroxylated fatty acid or dihydroxylated fatty acid composition comprising, as an active ingredient, a 9-lipoxygenase variant in which valine (V), the 569th amino acid, is substituted with phenylalanine (F) from the imanoic acid sequence of the 9-lipoxygenase. , The hydroxyl fatty acid or dihydroxyl fatty acid is 9-hydroxyarachidonic acid (9-hydroxyarachidonic acid), 9,15-dihydroxyarachidonic acid (9,15-dihydroxyarachidonic acid), 9-hydroxyeicosapentaenoic acid acid), 9,15-dihydroxyeicopentaenoic acid (9,15-dihydroxyeicopentaenoic acid), 11-hydroxydocosapentaenoic acid (11-hydroxydocosapentaenoic acid), 11,17-dihydroxyeicosapentaenoic acid (11 , 17-dihydroxydocosapentaenoic acid), 11-hydroxydocosahexaenoic acid (11-hydroxydocosahexaenoic acid) and 11,17-dihydroxydocosahexaenoic acid (11,17-dihydroxydocosahexaenoic acid). Or it provides a composition for producing dihydrogenated fatty acid.

상기 9-리폭시게나아제는 서열번호 1의 아미노산 서열로 이루어지고, 상기 9-리폭시게나아제 변이체는 서열번호 2의 아미노산 서열로 이루어진 것일 수 있다. The 9-lipoxygenase may consist of the amino acid sequence of SEQ ID NO: 1, and the 9-lipoxygenase variant may consist of the amino acid sequence of SEQ ID NO: 2.

상기 조성물은 아라키돈산(arachidonic acid), 에이코사펜타엔산(eicosapentaenoic acid), 도코사펜타엔산(eicosapentaenoic acid) 및 도코사헥사엔산(docosahexaenoic acid)으로 이루어진 군으로부터 선택된 하나 이상의 탄소수가 20~22개인 불포화 지방산을 포함하는 기질에 처리하기 위한 것일 수 있다. The composition has at least one carbon atom selected from the group consisting of arachidonic acid, eicosapentaenoic acid, eicosapentaenoic acid, and docosahexaenoic acid having 20 to 10 carbon atoms. It may be for treating substrates containing 22 unsaturated fatty acids.

본 발명의 일 구현예로, 서열번호 3의 염기 서열로 이루어진 9-리폭시게나아제 유전자; 또는 서열번호 4의 염기 서열로 이루어진 9-리폭시게나아제 변이체 유전자를 유효성분으로 포함하는 상기 수산화지방산 또는 이수산화지방산 제조용 조성물을 제공한다. In one embodiment of the present invention, the 9-lipoxygenase gene consisting of the nucleotide sequence of SEQ ID NO: 3; Alternatively, the composition for producing hydroxylated fatty acid or dihydroxidized fatty acid comprising the 9-lipoxygenase variant gene consisting of the nucleotide sequence of SEQ ID NO: 4 as an active ingredient is provided.

본 발명의 다른 구현예로, 상기 조성물을 기질에 처리하는 단계를 포함하는 상기 수산화지방산 또는 이수산화지방산 제조방법을 제공한다. In another embodiment of the present invention, the method for producing the hydroxylated fatty acid or the dihydrogenated fatty acid comprising the step of treating a substrate with the composition is provided.

상기 기질은 아라키돈산(arachidonic acid), 에이코사펜타엔산(eicosapentaenoic acid), 도코사펜타엔산(eicosapentaenoic acid) 및 도코사헥사엔산(docosahexaenoic acid)으로 이루어진 군으로부터 선택된 하나 이상의 탄소수가 20~22개인 불포화 지방산을 포함할 수 있다. The substrate has 20 to 10 carbon atoms selected from the group consisting of arachidonic acid, eicosapentaenoic acid, eicosapentaenoic acid and docosahexaenoic acid. 22 unsaturated fatty acids.

상기 처리는 pH 6.5~9.0 및 온도 20~40℃에서 수행되는 것일 수 있다. The treatment may be performed at a pH of 6.5 to 9.0 and a temperature of 20 to 40 °C.

본 발명의 또 다른 구현예로, 서열번호 3의 염기 서열로 이루어진 9-리폭시게나아제 유전자; 또는 서열번호 4의 염기 서열로 이루어진 9-리폭시게나아제 변이체 유전자를 포함하는 상기 수산화지방산 또는 이수산화지방산 제조용 재조합 발현 벡터를 제공한다. In another embodiment of the present invention, the 9-lipoxygenase gene consisting of the nucleotide sequence of SEQ ID NO: 3; Alternatively, a recombinant expression vector for preparing the hydroxylated fatty acid or dihydroxylated fatty acid containing the 9-lipoxygenase variant gene consisting of the nucleotide sequence of SEQ ID NO: 4 is provided.

본 발명의 또 다른 구현예로, 숙주세포에 상기 재조합 발현 벡터가 형질전환된 형질전환체를 제공한다. In another embodiment of the present invention, a transformant in which the recombinant expression vector is transformed into a host cell is provided.

본 발명의 또 다른 구현예로, 하기 화학식 1 내지 화학식 4 중 어느 하나로 표시되는, 신규 수산화지방산을 제공한다:In another embodiment of the present invention, a novel hydroxyl fatty acid represented by any one of Formulas 1 to 4 is provided:

[화학식 1][Formula 1]

Figure 112022054327526-pat00001
,
Figure 112022054327526-pat00001
,

[화학식 2][Formula 2]

Figure 112022054327526-pat00002
,
Figure 112022054327526-pat00002
,

[화학식 3][Formula 3]

Figure 112022054327526-pat00003
,
Figure 112022054327526-pat00003
,

[화학식 4][Formula 4]

Figure 112022054327526-pat00004
.
Figure 112022054327526-pat00004
.

본 발명의 또 다른 구현예로, 하기 화학식 5 내지 화학식 8 중 어느 하나로 표시되는, 신규 이수산화지방산을 제공한다:In another embodiment of the present invention, a novel dihydrogenated fatty acid represented by any one of Formulas 5 to 8 is provided:

[화학식 5][Formula 5]

Figure 112022054327526-pat00005
,
Figure 112022054327526-pat00005
,

[화학식 6][Formula 6]

Figure 112022054327526-pat00006
,
Figure 112022054327526-pat00006
,

[화학식 7][Formula 7]

Figure 112022054327526-pat00007
,
Figure 112022054327526-pat00007
,

[화학식 8][Formula 8]

Figure 112022054327526-pat00008
.
Figure 112022054327526-pat00008
.

본 발명에 따르면, 신규 미생물 유래 9-리폭시게나아제를 이용하여 생물전환 공정으로 신규 수산화지방산 또는 이수산화지방산을 높은 생산성과 높은 수율로 생산할 수 있으므로, 의약, 식품 및 화장품 등 다양한 산업 분야에서 유용하게 사용될 수 있을 것으로 기대된다.According to the present invention, novel hydroxylated fatty acids or dihydroxated fatty acids can be produced with high productivity and high yield through a bioconversion process using 9-lipoxygenase derived from novel microorganisms, so that they are useful in various industrial fields such as medicine, food, and cosmetics. expected to be usable.

본 발명에 따라 제조된 신규 수산화지방산 또는 이수산화지방산은 신호전달물질로서, 인간을 포함한 동물 내에서 다양한 생리활성 기능에 관여할 것으로 기대된다.The novel hydroxylated fatty acid or dihydroxated fatty acid prepared according to the present invention is a signal transducer and is expected to be involved in various physiologically active functions in animals including humans.

도 1은 본 발명의 9-리폭시게나아제를 이용하여 아라키돈산을 기질로 하여 생산되는 9-수산화아라키돈산 및 9,15-이수산화아라키돈산의 합성경로를 나타낸 것이다.
도 2는 9-수산화아라키돈산 및 9,15-이수산화아라키돈산의 생산을 확인한 HPLC 크로마토그램이다.
도 3은 9,15-이수산화아라키돈산의 물질동정을 위해 1D NMR을 진행한 결과이다.
도 4는 본 발명의 9-리폭시게나아제를 이용하여 에이코사펜타엔산을 기질로 하여 생산되는 9-수산화에이코사펜타엔산 및 9,15-이수산화에이코사펜타엔산의 합성경로를 나타낸 것이다.
도 5는 9-수산화에이코사펜타엔산 및 9,15-이수산화에이코사펜타엔산의 생산을 확인한 HPLC 크로마토그램이다.
도 6은 9,15-이수산화에이코사펜타엔산의 물질동정을 위해 1D NMR을 진행한 결과이다.
도 7은 본 발명의 9-리폭시게나아제를 이용하여 도코사펜타엔산을 기질로 하여 생산되는 11-수산화도코사펜타엔산 및 11,17-이수산화도코사펜타엔산의 합성경로를 나타낸 것이다.
도 8은 11-수산화도코사펜타엔산 및 11,17-이수산화도코사펜타엔산의 생산을 확인한 HPLC 크로마토그램이다.
도 9은 11,17-이수산화도코사펜타엔산의 물질동정을 위해 1D NMR을 진행한 결과이다.
도 10은 본 발명의 9-리폭시게나아제를 이용하여 도코사헥사엔산을 기질로 하여 생산되는 11-수산화도코사헥사엔산 및 11,17-이수산화도코사헥사엔산의 합성경로를 나타낸 것이다.
도 11은 11-수산화도코사헥사엔산 및 11,17-이수산화도코사헥사엔산의 생산을 확인한 HPLC 크로마토그램이다.
도 12는 11,17-이수산화도코사헥사엔산의 물질동정을 위해 1D NMR을 진행한 결과이다.
도 13a는 본 발명의 9-리폭시게나아제의 pH가 9-수산화아라키돈산, 9,15-이수산화아라키돈산 생산 활성에 미치는 영향을 나타낸 것이고(●: HEPES buffer, ▼: HEPPS buffer, ■: CHES buffer), 도 13b는 본 발명의 9-리폭시게나아제의 온도가 9-수산화아라키돈산, 9,15-이수산화아라키돈산 생산 활성에 미치는 영향을 나타낸 것이다.
도 14는 본 발명의 9-리폭시게나아제 변이체(V569F)를 이용한 아라키돈산으로부터 9-수산화아라키돈산의 시간별 생산량을 나타낸 것이다(■: 아라키돈산, ▼: 9-수산화아라키돈산, ●: 9,15-이수산화아라키돈산).
도 15는 본 발명의 9-리폭시게나아제를 이용한 아라키돈산으로부터 9,15-이수산화아라키돈산의 시간별 생산량을 나타낸 것이다(■: 아라키돈산, ▼: 9-수산화아라키돈산, ●: 9,15-이수산화아라키돈산).
도 16은 본 발명의 9-리폭시게나아제 변이체(V569F)를 이용한 에이코사펜타엔산으로부터 9-수산화에이코사펜타엔산의 시간별 생산량을 나타낸 것이다(■: 아라키돈산, ▼: 9-수산화에이코사펜타엔산, ●: 9,15-이수산화에이코사펜타엔산).
도 17은 본 발명의 9-리폭시게나아제를 이용한 에이코사펜타엔산으로부터 9,15-이수산화에이코사펜타엔산의 시간별 생산량을 나타낸 것이다(■: 에이코사펜타엔산, ▼: 9-수산화에이코사펜타엔산, ●: 9,15-이수산화에이코사펜타엔산).
도 18은 본 발명의 9-리폭시게나아제 변이체(V569F)를 이용한 도코사펜타엔산으로부터 11-수산화도코사펜타엔산의 시간별 생산량을 나타낸 것이다(■: 아라키돈산, ▼: 11-수산화도코사펜타엔산, ●: 11,17-이수산화도코사펜타엔산).
도 19는 본 발명의 9-리폭시게나아제를 이용한 도코사펜타엔산으로부터 11,17-이수산화도코사펜타엔산의 시간별 생산량을 나타낸 것이다(■: 도코사펜타엔산, ▼: 11-수산화도코사펜타엔산, ●: 11,17-이수산화도코사펜타엔산).
도 20은 본 발명의 9-리폭시게나아제 변이체(V569F)를 이용한 도코사헥사엔산으로부터 11-수산화도코사헥사엔산의 시간별 생산량을 나타낸 것이다(■: 아라키돈산, ▼: 11-수산화도코사헥사엔산, ●: 11,17-이수산화도코사헥사엔산).
도 21은 본 발명의 9-리폭시게나아제를 이용한 도코사헥사엔산으로부터 11,17-이수산화도코사헥사엔산의 시간별 생산량을 나타낸 것이다(■: 도코사헥사엔산, ▼: 11-수산화도코사헥사엔산, ●: 11,17-이수산화도코사헥사엔산).
도 22는 본 발명의 9-리폭시게나아제를 합성시킨 재조합 벡터를 나타낸 것이다.Figure 1 shows the synthesis pathway of 9-hydroxyarachidonic acid and 9,15-arachidonic acid dihydroxide produced using arachidonic acid as a substrate using the 9-lipoxygenase of the present invention.
Figure 2 is an HPLC chromatogram confirming the production of 9-hydroxyarachidonic acid and 9,15-arachidonic acid dihydroxide.
3 is a result of 1D NMR for material identification of 9,15-arachidonic acid dihydroxide.
Figure 4 shows the synthesis pathway of 9-hydroxyeicosapentaenoic acid and 9,15-dihydroxyeicosapentaenoic acid produced using eicosapentaenoic acid as a substrate using the 9-lipoxygenase of the present invention. will be.
5 is an HPLC chromatogram confirming the production of 9-hydroxyeicosapentaenoic acid and 9,15-dihydroxyeicosapentaenoic acid.
6 is a result of 1D NMR for material identification of 9,15-dihydroxyeicosapentaenoic acid.
7 shows the synthesis pathway of 11-hydroxydocosapentaenoic acid and 11,17-dihydroxydocosapentaenoic acid produced using docosapentaenoic acid as a substrate using the 9-lipoxygenase of the present invention. .
8 is an HPLC chromatogram confirming the production of 11-hydroxydocosapentaenoic acid and 11,17-dihydroxydocosapentaenoic acid.
9 is a result of 1D NMR for material identification of 11,17-dihydroxydocosapentaenoic acid.
Figure 10 shows the synthesis pathway of 11-hydroxydocosahexaenoic acid and 11,17-dihydroxide docosahexaenoic acid produced using docosahexaenoic acid as a substrate using the 9-lipoxygenase of the present invention. .
11 is an HPLC chromatogram confirming the production of 11-hydroxydocosahexaenoic acid and 11,17-dihydroxydocosahexaenoic acid.
12 is a result of 1D NMR for material identification of 11,17-dihydroxide docosahexaenoic acid.
Figure 13a shows the effect of the pH of the 9-lipoxygenase of the present invention on the production activity of 9-hydroxyarachidonic acid and 9,15-arachidonic acid dihydroxide (●: HEPES buffer, ▼: HEPPS buffer, ■: CHES buffer), Figure 13b shows the effect of the temperature of the 9-lipoxygenase of the present invention on the production activity of 9-hydroxyarachidonic acid and 9,15-arachidonic acid dihydroxide.
Figure 14 shows the hourly production of 9-hydroxyarachidonic acid from arachidonic acid using the 9-lipoxygenase variant (V569F) of the present invention (■: arachidonic acid, ▼: 9-hydroxyarachidonic acid, ●: 9,15 -arachidonic acid dihydroxide).
Figure 15 shows the hourly production of 9,15-arachidonic acid dihydroxide from arachidonic acid using the 9-lipoxygenase of the present invention (■: arachidonic acid, ▼: 9-arachidonic acid hydroxyl, ●: 9,15- arachidonic acid dihydroxide).
Figure 16 shows the production of 9-hydroxyeicosapentaenoic acid over time from eicosapentaenoic acid using the 9-lipoxygenase variant (V569F) of the present invention (■: arachidonic acid, ▼: 9-eicosa hydroxylation) Pentaenoic acid, ●: 9,15-dihydroxyeicosapentaenoic acid).
Figure 17 shows the hourly production of 9,15-dihydroxed eicosapentaenoic acid from eicosapentaenoic acid using 9-lipoxygenase of the present invention (■: eicosapentaenoic acid, ▼: 9-hydroxylated eicosapentaenoic acid, ●: 9,15-dihydroxyeicosapentaenoic acid).
Figure 18 shows the hourly production of 11-hydroxydocosapentaenoic acid from docosapentaenoic acid using the 9-lipoxygenase variant (V569F) of the present invention (■: arachidonic acid, ▼: 11-hydroxydocosa Pentaenoic acid, ●: 11,17-dihydroxydocosapentaenoic acid).
Figure 19 shows the hourly production of 11,17-dihydroxide docosapentaenoic acid from docosapentaenoic acid using 9-lipoxygenase of the present invention (■: docosapentaenoic acid, ▼: 11-hydroxylation degree Cosapentaenoic acid, ●: 11,17-dihydroxydocosapentaenoic acid).
Figure 20 shows the hourly production of 11-hydroxydocosahexaenoic acid from docosahexaenoic acid using the 9-lipoxygenase variant (V569F) of the present invention (■: arachidonic acid, ▼: 11-hydroxydocosa Hexaenoic acid, ●: 11,17-dihydrodocosahexaenoic acid).
Figure 21 shows the hourly production of 11,17-dihydroxide docosahexaenoic acid from docosahexaenoic acid using the 9-lipoxygenase of the present invention (■: docosahexaenoic acid, ▼: 11-hydroxylation degree cosahexaenoic acid, ●: 11,17-dihydroxydocosahexaenoic acid).
22 shows a recombinant vector synthesized with the 9-lipoxygenase of the present invention.

본 발명자들은 최초로 그람-음성균에 해당하는 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus)로부터 9-리폭시게나아제의 유전자 및 효소를 동정하였다. 이후, 본 발명자들은 이를 이용하여 생물전환 공정을 통해 보다 효과적으로 수산화지방산 및 이수산화지방산을 생산하기 위한 지속적인 연구를 수행하였다. The present inventors first identified the gene and enzyme of 9-lipoxygenase from Sphingomonas macrogoltabidus , which is a Gram-negative bacterium. Then, the present inventors conducted continuous research to more effectively produce hydroxylated fatty acids and dihydroxated fatty acids through a bioconversion process using the same.

구체적으로, 본 발명자들은 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) DSM 8826 유래 9-리폭시게나아제를 클로닝하여 재조합 발현 벡터 및 이로부터 형질전환된 미생물을 제작하고, 이를 이용하여 전세포를 생산한 다음, 이를 기질에 처리함으로써 이러한 친환경적인 과정으로 수산화지방산 및 이수산화지방산을 높은 생산성과 높은 수율로 생산할 수 있음을 확인하고, 본 발명을 완성하였다.Specifically, the present inventors clone Sphingomonas macrogoltabidus DSM 8826-derived 9-lipoxygenase to construct a recombinant expression vector and a microorganism transformed therefrom, and use this to produce whole cells Next, it was confirmed that hydroxyl fatty acids and dihydroxidated fatty acids could be produced with high productivity and high yield through such an environmentally friendly process by treating them with substrates, and completed the present invention.

이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 명세서 내 "수산화지방산"이라 함은 지방산, 바람직하게, 탄소수가 20~22개인 불포화 지방산에 1개의 수산기가 치환된 것을 의미하는 것으로, 구체적으로, 9-수산화아라키돈산(9-hydroxyarachidonic acid; 9-HETE), 9-수산화에이코사펜타엔산(9-hydroxyeicosapentaenoic acid; 9-HEPE), 11-수산화도코사펜타엔산(11-hydroxydocosapentaenoic acid; 11-HDOPE) 및 11-수산화도코사헥사엔산(11-hydroxydocosahexaenoic acid; 11-HDOHE)로 이루어진 군으로부터 선택된 어느 하나 이상일 수 있고, 이들은 모두 신규 화합물로서, 순서대로 하기 화학식 1 내지 4로 표시될 수 있다: The term "hydroxyl fatty acid" in the present specification means that one hydroxyl group is substituted for a fatty acid, preferably, an unsaturated fatty acid having 20 to 22 carbon atoms, specifically, 9-hydroxyarachidonic acid (9-hydroxyarachidonic acid; 9 -HETE), 9-hydroxyeicosapentaenoic acid (9-HEPE), 11-hydroxydocosapentaenoic acid (11-HDOPE) and 11-hydroxydocosahexaenoic acid (11-hydroxydocosahexaenoic acid; 11-HDOHE), which may be any one or more selected from the group consisting of, all of which are novel compounds, which may be sequentially represented by Chemical Formulas 1 to 4:

[화학식 1][Formula 1]

Figure 112022054327526-pat00009
,
Figure 112022054327526-pat00009
,

[화학식 2][Formula 2]

Figure 112022054327526-pat00010
,
Figure 112022054327526-pat00010
,

[화학식 3][Formula 3]

Figure 112022054327526-pat00011
,
Figure 112022054327526-pat00011
,

[화학식 4][Formula 4]

Figure 112022054327526-pat00012
.
Figure 112022054327526-pat00012
.

본 명세서 내 "이수산화지방산"이라 함은 지방산, 바람직하게, 탄소수가 20~22개인 불포화 지방산에 2개의 수산기가 치환된 것을 의미하는 것으로, 구체적으로, 9,15-이수산화아라키돈산(9,15-dihydroxyarachidonic acid; 9,15-diHETE), 9,15-이수산화에이코사펜타엔산(9,15-dihydroxyeicosapentaenoic acid; 9,15-diHEPE), 11,17-이수산화도코사펜타엔산(11,17-dihydroxydocosapentaenoic acid; 11,17-diHDOPE) 및 11,17-이수산화도코사헥사엔산(11,17-dihydroxydocosahexaenoic acid; 11,17-diHDOHE)로 이루어진 군으로부터 선택된 하나 이상일 수 있고, 이들은 모두 신규 화합물로서, 순서대로 하기 화학식 5 내지 8로 표시될 수 있다: The term "dihydroxylated fatty acid" in the present specification means a fatty acid, preferably, an unsaturated fatty acid having 20 to 22 carbon atoms in which two hydroxyl groups are substituted, specifically, 9,15-dihydroxarachidonic acid (9, 15-dihydroxyarachidonic acid; 9,15-diHETE), 9,15-dihydroxyeicosapentaenoic acid (9,15-dihydroxyeicosapentaenoic acid; 9,15-diHEPE), 11,17-dihydroxyeicosapentaenoic acid (11 ,17-dihydroxydocosapentaenoic acid; 11,17-diHDOPE) and 11,17-dihydroxydocosahexaenoic acid; may be at least one selected from the group consisting of (11,17-dihydroxydocosahexaenoic acid; 11,17-diHDOHE), all of which are novel As a compound, it can be represented by the following formulas 5 to 8 in order:

[화학식 5][Formula 5]

Figure 112022054327526-pat00013
,
Figure 112022054327526-pat00013
,

[화학식 6][Formula 6]

Figure 112022054327526-pat00014
,
Figure 112022054327526-pat00014
,

[화학식 7][Formula 7]

Figure 112022054327526-pat00015
,
Figure 112022054327526-pat00015
,

[화학식 8][Formula 8]

Figure 112022054327526-pat00016
.
Figure 112022054327526-pat00016
.

수산화지방산 또는 이수산화지방산 제조용 조성물A composition for preparing hydroxylated fatty acid or dihydroxated fatty acid

본 발명은 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) 유래 9-리폭시게나아제; 또는 상기 9-리폭시게나아제의 이마노산 서열로부터, 569번째 아미노산인 발린(V)을 페닐알라닌(F)로 치환시킨 9-리폭시게나아제 변이체를 유효성분으로 포함하는 상기 수산화지방산 또는 이수산화지방산 제조용 조성물을 제공한다. The present invention is Sphingomonas macrogoltabidus ( Sphingomonas macrogoltabidus ) Derived 9-lipoxygenase; or a 9-lipoxygenase variant in which valine (V), the 569th amino acid from the imanoic acid sequence of the 9-lipoxygenase is substituted with phenylalanine (F), as an active ingredient. provides

본 발명에 따른 수산화지방산 또는 이수산화지방산 제조용 조성물은 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) 유래 9-리폭시게나아제; 또는 상기 9-리폭시게나아제의 이마노산 서열로부터, 569번째 아미노산인 발린(V)을 페닐알라닌(F)로 치환시킨 9-리폭시게나아제 변이체를 유효성분으로 포함하는 것으로, 상기 9-리폭시게나아제 또는 상기 9-리폭시게나아제 변이체를 포함하는 전세포를 포함할 수 있다.The composition for producing hydroxylated fatty acid or dihydroxidized fatty acid according to the present invention includes Sphingomonas macrogoltabidus 9-lipoxygenase derived from Sphingomonas macrogoltabidus ; or a 9-lipoxygenase variant in which valine (V), the 569th amino acid from the imanoic acid sequence of the 9-lipoxygenase is substituted with phenylalanine (F) as an active ingredient, the 9-lipoxygenase or Whole cells containing the 9-lipoxygenase variant may be included.

먼저, 상기 9-리폭시게나아제는 야생형(wild-type)으로서, 이를 이용하여 지방산으로부터 수산화지방산 또는 이수산화지방산을 생산할 수 있는데, 구체적으로, 지방산으로부터 산화반응을 통해 수산화지방산을 생산한 다음, 추가 산화반응을 통해 이수산화지방산을 생산하는 2단계 산화반응을 잘 수행할 수 있다. 따라서, 이수산화지방산이 주생성물이고, 수산화지방산이 부산물일 수 있다. 이때, 이수산화지방산은 탄소수 20개의 불포화 지방산의 9번의 탄소 위치에 수산기가 형성된 후, 15번의 탄소 위치에 수산기가 형성된 것일 수 있고, 혹은 탄소수 22개 이상의 불포화 지방산의 11번 탄소 위치에 수산기가 형성된 후, 17번의 탄소 위치에 수산기가 형성된 것일 수 있다. First, the 9-lipoxygenase is wild-type, which can be used to produce hydroxylated fatty acid or dihydrogenated fatty acid from fatty acids. Specifically, hydroxylated fatty acids are produced from fatty acids through an oxidation reaction, and then added. It can perform well the two-step oxidation reaction to produce dihydrogenated fatty acid through oxidation reaction. Therefore, dihydroxated fatty acid may be the main product and hydroxylated fatty acid may be a by-product. At this time, the dihydroxy fatty acid may be one in which a hydroxyl group is formed at the 9th carbon position of an unsaturated fatty acid having 20 carbon atoms and then a hydroxyl group is formed at the 15th carbon position, or a hydroxyl group is formed at the 11th carbon position of an unsaturated fatty acid having 22 or more carbon atoms. After that, a hydroxyl group may be formed at the position of carbon number 17.

또한, 상기 9-리폭시게나아제는 서열번호 1의 아미노산 서열로 이루어진 것뿐만 아니라, 이의 기능적 동등물, 즉, 상기 서열에 하나 이상의 치환, 결손 등의 돌연변이를 유발하여 본 발명의 목적을 달성하는 모든 돌연변이체를 포함하는 것을 의미하며, 상기 9-리폭시게나아제는 서열번호 3의 염기서열로 이루어진 유전자로부터 발현된 산물일 수 있다.In addition, the 9-lipoxygenase is not only composed of the amino acid sequence of SEQ ID NO: 1, but also a functional equivalent thereof, that is, all substances that achieve the object of the present invention by inducing mutations such as one or more substitutions or deletions in the sequence. It means including a mutant, and the 9-lipoxygenase may be a product expressed from a gene consisting of the nucleotide sequence of SEQ ID NO: 3.

다음으로, 상기 9-리폭시게나아제 변이체는 상기 9-리폭시게나아제가 위치 지정 돌연변이에 의해 변형된 것으로서, 이를 이용하여 지방산으로부터 수산화지방산 또는 이수산화지방산을 생산할 수 있는데, 구체적으로, 지방산으로부터 산화반응을 통해 수산화지방산을 생산하는 1단계 산화반응을 잘 수행할 수 있다. 따라서, 수산화지방산이 주생성물이고, 이수산화지방산이 부산물일 수 있다. 이때, 수산화지방산은 탄소수 20개의 불포화 지방산의 9번의 탄소 위치에 수산기가 형성된 것일 수 있고, 탄소수 22개 이상의 불포화 지방산의 11번의 탄소 위치에 수산기가 형성된 것일 수 있다. Next, the 9-lipoxygenase variant is one in which the 9-lipoxygenase is modified by site-directed mutation, and hydroxylated fatty acid or dihydrogenated fatty acid can be produced from fatty acid using this variant. Specifically, oxidation reaction from fatty acid Through this, the first-step oxidation reaction to produce hydroxylated fatty acids can be performed well. Therefore, hydroxylated fatty acid may be the main product, and dihydrogenated fatty acid may be a by-product. In this case, the hydroxyl fatty acid may be one in which a hydroxyl group is formed at the 9th carbon position of an unsaturated fatty acid having 20 carbon atoms, or a hydroxyl group formed at the 11th carbon position of an unsaturated fatty acid having 22 or more carbon atoms.

또한, 상기 9-리폭시게나아제 변이체는 서열번호 2의 아미노산 서열로 이루어진 것뿐만 아니라, 이의 기능적 동등물, 즉, 상기 서열에 하나 이상의 치환, 결손 등의 돌연변이를 유발하여 본 발명의 목적을 달성하는 모든 돌연변이체를 포함하는 것을 의미하며, 상기 9-리폭시게나아제 변이체는 서열번호 3의 염기서열로 이루어진 유전자로부터 발현된 산물일 수 있다.In addition, the 9-lipoxygenase variant is not only composed of the amino acid sequence of SEQ ID NO: 2, but also a functional equivalent thereof, that is, by inducing mutations such as one or more substitutions or deletions in the sequence to achieve the object of the present invention It means including all mutants, and the 9-lipoxygenase mutant may be a product expressed from a gene consisting of the nucleotide sequence of SEQ ID NO: 3.

한편, 상기 9-리폭시게나아제 또는 9-리폭시게나아제 변이체의 최적 활성 pH는 6.5 내지 9.0, 바람직하게 8.0 내지 9.0이고, 최적 활성 온도는 20℃ 내지 40℃, 바람직하게, 25℃ 내지 35℃이다. On the other hand, the optimal activity pH of the 9-lipoxygenase or 9-lipoxygenase variant is 6.5 to 9.0, preferably 8.0 to 9.0, and the optimal activity temperature is 20 ° C to 40 ° C, preferably 25 ° C to 35 ° C. .

구체적으로, 상기 9-리폭시게나아제 또는 상기 9-리폭시게나아제 변이체를 포함하는 전세포는 서열번호 1 또는 2의 아미노산 서열을 암호화하는 유전자 또는 서열번호 3 또는 4의 염기서열로 이루어진 유전자를 포함하는 재조합 발현 벡터로 형질전환된 형질전환 미생물을 배양하고, 이를 수득하여 사용하는 것이 바람직하다.Specifically, the whole cell containing the 9-lipoxygenase or the 9-lipoxygenase variant comprises a gene encoding the amino acid sequence of SEQ ID NO: 1 or 2 or a gene consisting of the nucleotide sequence of SEQ ID NO: 3 or 4 It is preferable to culture a transformed microorganism transformed with a recombinant expression vector, obtain it, and use it.

상기 재조합 발현 벡터로서, 유전자 재조합을 위하여 당업계에서 사용되고 있는 플라스미드 벡터라면 어느 벡터를 사용해도 무방하고, 구체적으로 pET-28a-c(+) 벡터를 사용하는 것이 보다 바람직하나, 이에 한정되지 않는다.As the recombinant expression vector, any vector may be used as long as it is a plasmid vector used in the art for genetic recombination, and it is more preferable to use the pET-28a-c(+) vector, but is not limited thereto.

상기 형질전환 미생물로서, 재조합 벡터로 형질전환하여 목적하는 단백질을 과발현하는 시스템으로 당업계에 사용되고 있는 미생물이라면 어느 미생물을 사용해도 무방하고, 구체적으로 대장균 ER 2566 균주를 사용하는 것이 보다 바람직하나, 이에 한정되지 않는다.As the transformed microorganism, any microorganism may be used as long as it is a microorganism used in the art as a system for overexpressing a desired protein by transforming with a recombinant vector, and specifically, it is more preferable to use E. coli ER 2566 strain, but Not limited.

상기 전세포는 ⅰ) 상기 미생물의 배양액을 원심분리하여 1차 전세포를 회수하는 단계; ⅱ) 상기 회수한 전세포를 생리식염수(saline solution)으로 세척하는 단계; ⅲ) 상기 세척된 전세포를 2차 원심분리하여 상등액을 제거하고 전세포를 얻는 단계; 및 ⅳ) 상기 2차로 회수한 전세포를 다시 한번 생리식염수로 세척하는 단계를 포함하여 수득될 수 있다. 구체적으로, ⅰ) 단계에서 전세포의 회수는 원심분리기 등 당업계 공지된 기기를 사용하여 6,000xg 내외의 범위에서 수행될 수 있고, ⅱ) 단계에서 전세포의 세척은 0.85% 이하의 염화나트륨 용액으로 수행하는 것이 적당하다.The whole cells may be prepared by: i) recovering primary whole cells by centrifuging the microbial culture medium; ii) washing the recovered whole cells with saline solution; iii) removing the supernatant by second centrifugation of the washed whole cells and obtaining whole cells; and iv) washing the secondly recovered whole cells with physiological saline once again. Specifically, the recovery of the whole cells in step i) can be performed in the range of around 6,000xg using a device known in the art such as a centrifuge, and the washing of the whole cells in step ii) is done with a sodium chloride solution of 0.85% or less. it is appropriate to do

이때, 상기 조성물 내 상기 리폭시게나아제 변이체를 포함하는 전세포의 농도는 0.01 g/L 내지 1 g/L인 것이 바람직하고, 0.05 g/L 내지 0.5 g/L인 것이 보다 바람직하나, 이에 한정되지 않는다.At this time, the concentration of whole cells containing the lipoxygenase variant in the composition is preferably 0.01 g/L to 1 g/L, more preferably 0.05 g/L to 0.5 g/L, but is not limited thereto. don't

상기 조성물은 아라키돈산(arachidonic acid), 에이코사펜타엔산(eicosapentaenoic acid), 도코사펜타엔산(eicosapentaenoic acid) 및 도코사헥사엔산(docosahexaenoic acid)으로 이루어진 군으로부터 선택된 하나 이상의 탄소수가 20~22개인 불포화 지방산(바람직하게, 하나 이상의 cis, cis-1,4 펜타디엔을 가지는)을 포함하는 기질에 처리하기 위한 것일 수 있다.The composition has at least one carbon atom selected from the group consisting of arachidonic acid, eicosapentaenoic acid, eicosapentaenoic acid, and docosahexaenoic acid having 20 to 10 carbon atoms. 22 unsaturated fatty acids (preferably with at least one cis, cis-1,4 pentadiene).

상기 기질 내 상기 탄소수가 20~22개인 불포화 지방산의 농도는 0.1 mM 내지 10 mM인 것이 바람직하나, 이에 한정되지 않는다. 상기 탄소수가 20~22개인 불포화 지방산은 이러한 농도를 유지함으로써, 수산화지방산 또는 이수산화지방산의 생산 농도를 향상시킬 수 있다.The concentration of the unsaturated fatty acid having 20 to 22 carbon atoms in the substrate is preferably 0.1 mM to 10 mM, but is not limited thereto. By maintaining the concentration of the unsaturated fatty acid having 20 to 22 carbon atoms, the production concentration of hydroxyl fatty acid or dihydroxyl fatty acid can be improved.

또한, 본 발명은 서열번호 3의 염기 서열로 이루어진 9-리폭시게나아제 유전자; 또는 서열번호 4의 염기 서열로 이루어진 9-리폭시게나아제 변이체 유전자를 유효성분으로 포함하는 상기 수산화지방산 또는 이수산화지방산 제조용 조성물을 제공한다.In addition, the present invention is a 9-lipoxygenase gene consisting of the nucleotide sequence of SEQ ID NO: 3; Alternatively, the composition for producing hydroxylated fatty acid or dihydroxidized fatty acid comprising the 9-lipoxygenase variant gene consisting of the nucleotide sequence of SEQ ID NO: 4 as an active ingredient is provided.

뿐만 아니라, 본 발명은 서열번호 3의 염기 서열로 이루어진 9-리폭시게나아제 유전자; 또는 서열번호 4의 염기 서열로 이루어진 9-리폭시게나아제 변이체 유전자를 포함하는 상기 수산화지방산 또는 이수산화지방산 제조용 재조합 발현 벡터와, 숙주 세포에 상기 재조합 발현 벡터가 형질전환된 형질전환체를 제공한다.In addition, the present invention is a 9-lipoxygenase gene consisting of the nucleotide sequence of SEQ ID NO: 3; Alternatively, the recombinant expression vector for preparing the hydroxyl fatty acid or dihydroxyl fatty acid containing the 9-lipoxygenase mutant gene consisting of the nucleotide sequence of SEQ ID NO: 4 and the transformant transformed with the recombinant expression vector in a host cell are provided.

먼저, 상기 리폭시게나아제 유전자는 서열번호 3의 염기 서열로 이루어진 것뿐만 아니라, 이의 기능적 동등물, 즉, 상기 서열에 하나 이상의 치환, 결손 등의 돌연변이를 유발하여 본 발명의 목적을 달성하는 모든 돌연변이체를 포함하는 것을 의미한다. First, the lipoxygenase gene is not only composed of the nucleotide sequence of SEQ ID NO: 3, but also a functional equivalent thereof, that is, all mutations that achieve the object of the present invention by inducing mutations such as one or more substitutions or deletions in the sequence. It means to include the body.

다음으로, 상기 리폭시게나아제 변이체 유전자는 서열번호 4의 염기 서열로 이루어진 것뿐만 아니라, 이의 기능적 동등물, 즉, 상기 서열에 하나 이상의 치환, 결손 등의 돌연변이를 유발하여 본 발명의 목적을 달성하는 모든 돌연변이체를 포함하는 것을 의미한다. Next, the lipoxygenase mutant gene is not only composed of the nucleotide sequence of SEQ ID NO: 4, but also a functional equivalent thereof, that is, by inducing mutations such as one or more substitutions, deletions, etc. in the sequence to achieve the object of the present invention It means to include all mutants.

수산화지방산 또는 이수산화지방산 제조방법Method for producing hydroxylated fatty acid or dihydrogenated fatty acid

본 발명은 상기 조성물을 기질에 처리하는 단계를 포함하는 상기 수산화지방산 또는 이수산화지방산 제조방법을 제공한다. The present invention provides a method for preparing the hydroxylated fatty acid or dihydrogenated fatty acid comprising the step of treating a substrate with the composition.

본 발명에 따른 수산화지방산 또는 이수산화지방산의 제조방법은 상기 조성물을 기질에 처리하는 단계를 포함한다. The method for producing hydroxylated fatty acid or dihydroxidized fatty acid according to the present invention includes the step of treating a substrate with the composition.

상기 조성물은 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) 유래 9-리폭시게나아제; 또는 상기 9-리폭시게나아제의 이마노산 서열로부터, 569번째 아미노산인 발린(V)을 페닐알라닌(F)로 치환시킨 9-리폭시게나아제 변이체를 유효성분으로 포함하거나, 서열번호 3의 염기 서열로 이루어진 9-리폭시게나아제 유전자; 또는 서열번호 4의 염기 서열로 이루어진 9-리폭시게나아제 변이체 유전자를 유효성분으로 포함하는 것으로, 구체적인 내용에 대해서는 전술한 바와 같다. The composition is Sphingomonas macrogoltabidus ( Sphingomonas macrogoltabidus ) Derived 9-lipoxygenase; or a 9-lipoxygenase variant in which valine (V), the 569th amino acid from the imanoic acid sequence of the 9-lipoxygenase is substituted with phenylalanine (F) as an active ingredient, or consisting of the nucleotide sequence of SEQ ID NO: 3 9-lipoxygenase gene; or a 9-lipoxygenase mutant gene consisting of the nucleotide sequence of SEQ ID NO: 4 as an active ingredient, and details are as described above.

상기 기질은 아라키돈산(arachidonic acid), 에이코사펜타엔산(eicosapentaenoic acid), 도코사펜타엔산(eicosapentaenoic acid) 및 도코사헥사엔산(docosahexaenoic acid)으로 이루어진 군으로부터 선택된 하나 이상의 탄소수가 20~22개인 불포화 지방산을 포함할 수 있다.The substrate has 20 to 10 carbon atoms selected from the group consisting of arachidonic acid, eicosapentaenoic acid, eicosapentaenoic acid and docosahexaenoic acid. 22 unsaturated fatty acids.

상기 기질 내 상기 탄소수가 20~22개인 불포화 지방산의 농도는 0.1 mM 내지 10 mM인 것이 바람직하나, 이에 한정되지 않는다. 상기 탄소수가 20~22개인 불포화 지방산은 이러한 농도를 유지함으로써, 수산화지방산 또는 이수산화지방산의 생산 농도를 향상시킬 수 있다.The concentration of the unsaturated fatty acid having 20 to 22 carbon atoms in the substrate is preferably 0.1 mM to 10 mM, but is not limited thereto. By maintaining the concentration of the unsaturated fatty acid having 20 to 22 carbon atoms, the production concentration of hydroxyl fatty acid or dihydroxyl fatty acid can be improved.

상기 처리는 pH 6.5~9.0 및 20~40℃의 온도에서 수행되는 것이 바람직하고, pH 8.0~9.0 및 25~35℃의 온도에서 수행되는 것이 보다 바람직하나, 이에 한정되지 않는다. 이러한 pH 조건을 유지하기 위해서 반응용매로 HEPES, EPPS 또는 CHES 완충용액을 사용할 수 있다. 이러한 pH 조건을 유지함으로써, 사용되는 효소를 최적으로 활성화시킬 수 있어, 최종적으로 수산화지방산 또는 이수산화지방산을 높은 생산성 및 높은 수율로 제조할 수 있다. 또한, 상기 처리는 10분 이상 수행되는 것이 바람직하나, 이에 한정되지 않는다.The treatment is preferably performed at pH 6.5 to 9.0 and a temperature of 20 to 40° C., and more preferably performed at pH 8.0 to 9.0 and a temperature of 25 to 35° C., but is not limited thereto. In order to maintain these pH conditions, HEPES, EPPS or CHES buffer may be used as a reaction solvent. By maintaining this pH condition, the enzyme used can be optimally activated, and finally, hydroxylated fatty acid or dihydroxated fatty acid can be produced with high productivity and high yield. In addition, the treatment is preferably performed for 10 minutes or longer, but is not limited thereto.

한편, 상기 처리는 100mM 내지 400mM 농도의 시스테인과 반응을 통해 수행될 수 있고, 100 mM 내지 300mM 농도의 시스테인과 반응을 통해 수행되는 것이 바람직하나, 이에 한정되지 않는다. 이로써, 사용되는 효소를 최적으로 활성화시킬 수 있어, 최종적으로 수산화지방산 또는 이수산화지방산을 높은 생산성 및 높은 수율로 제조할수 있다.Meanwhile, the treatment may be performed through a reaction with cysteine at a concentration of 100 mM to 400 mM, and preferably performed through a reaction with cysteine at a concentration of 100 mM to 300 mM, but is not limited thereto. In this way, the enzyme to be used can be optimally activated, and finally, hydroxylated fatty acid or dihydroxated fatty acid can be produced with high productivity and high yield.

상기한 바와 같이, 신규 미생물 유래 9-리폭시게나아제를 이용하여 생물전환 공정으로 신규 수산화지방산 또는 이수산화지방산을 높은 생산성과 높은 수율로 생산할 수 있으므로, 의약, 식품 및 화장품 등 다양한 산업 분야에서 유용하게 사용될 수 있을 것으로 기대된다. As described above, since 9-lipoxygenase derived from novel microorganisms can be used to produce novel hydroxylated fatty acids or dihydroxated fatty acids with high productivity and high yield through a bioconversion process, it is useful in various industrial fields such as medicine, food, and cosmetics. expected to be usable.

본 발명에 따라 제조된 신규 수산화지방산 또는 이수산화지방산은 신호전달물질로서, 인간을 포함한 동물 내에서 다양한 생리활성 기능에 관여할 것으로 기대된다.The novel hydroxylated fatty acid or dihydroxated fatty acid prepared according to the present invention is a signal transducer and is expected to be involved in various physiologically active functions in animals including humans.

이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시계는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

실시예 1. 9-리폭시게나아제의 발현을 위해 유전자를 포함하는 재조합 발현 벡터 및 미생물의 제작Example 1. Construction of recombinant expression vectors and microorganisms containing genes for the expression of 9-lipoxygenase

본 발명의 9-리폭시게나아제 유전자를 제조하기 위하여, 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) DSM 8826으로부터 유래한 9-리폭시게나아제 유전자를 먼저 분리하였다.To prepare the 9-lipoxygenase gene of the present invention, the 9-lipoxygenase gene derived from Sphingomonas macrogoltabidus DSM 8826 was first isolated.

구체적으로, 유전자 염기서열과 아미노산 사열이 이미 특정되어 있는 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) DSM 8826 DNA 염기서열을 기초로 하여 중합효소 연쇄반응(PCR)을 실시하기 위하여 스핑고모나스 마크로골 타비두스(Sphingomonas macrogoltabidus) DSM 8826 의 genomic DNA를 추출(Intron)하여 이를 PCR의 주형으로 사용하였으며, 9-리폭시게나아제의 DNA 염기서열을 기초로 한 프라이머(primer)를 각각 고안하여 중합효소 연쇄반응(PCR)을 실시하였다. 9-리폭시게나아제의 서열의 프라이머는 제한효소를 Nde I과 Hind III을 이용하였으며, 프라이머 서열은 F: GCT CGA CCA TAT GTC CTT TGT GTC GCC TTC GCT G와 R: GCG CCC AAG CTT TCA GAT ATT GAT GCT CGT CGG G로 구성하였다. 또한, 플라스미드 벡터 pET 28a(+) (Novagen사 제품)를 제한효소를 처리하여 준비하였으며, ligation하여 각각 pET 28a(+)/9-리폭시게나아제를 제작하였다.Specifically, to carry out polymerase chain reaction (PCR) based on the Sphingomonas macrogoltabidus DSM 8826 DNA sequence, in which the gene sequence and amino acid sequence have already been specified, Sphingomonas macrogol Tavidus ( Sphingomonas macrogoltabidus ) The genomic DNA of DSM 8826 was extracted (Intron) and used as a template for PCR, and primers based on the DNA sequence of 9-lipoxygenase were designed, respectively, and polymerase chain reaction was performed. (PCR) was performed. Nde I and Hind III were used as restriction enzymes for the primer sequence of 9-lipoxygenase, and the primer sequence was F: GCT CGA CCA TAT GTC CTT TGT GTC GCC TTC GCT G and R: GCG CCC AAG CTT TCA GAT ATT GAT It was composed of GCT CGT CGG G. In addition, the plasmid vector pET 28a(+) (manufactured by Novagen) was prepared by treatment with a restriction enzyme, and ligated to prepare pET 28a(+)/9-lipoxygenase, respectively.

상기와 같이 얻은 재조합 발현 벡터는 통상적인 형질전환 방법에 의하여 New England Biolabs (Hertfordshire, UK)에서 구매한 대장균 ER 2566 균주에 형질 전환하고, 상기 형질전환 된 미생물은 20% 글리세린(glycerine) 용액을 첨가하여 9-수산화지방산 및 9,15-이수산화지방산의 생산을 위한 배양을 실시하기 전에 -70℃에 냉동 보관하였다.The recombinant expression vector obtained as described above was transformed into an E. coli ER 2566 strain purchased from New England Biolabs (Hertfordshire, UK) by a conventional transformation method, and a 20% glycerine solution was added to the transformed microorganism. and stored frozen at -70 ° C. before culturing for the production of 9-hydroxyl fatty acid and 9,15-dihydroxide fatty acid.

실시예 2. 9-리폭시게나아제의 제조Example 2. Preparation of 9-lipoxygenase

효소의 단백질 발현을 위하여, 실시예 1에서 냉동 보관시킨 형질전환된 미생물들은 500 ml의 LB(Difco, Sparks, MD, USA) 배지와 20 μg/ml의 카나마이신을 가지는 플라스크에서 200 rev/min의 통기조건 하에서 37℃에서 배양하였다. 박테리아의 흡광도가 600 nm에서 0.6에서 0.8에 도달할 때, 효소들의 단백질 발현을 유도하기 위하여 최종농도 0.1 mM IPTG를 첨가한 후 그 배양액을 16-18시간 동안 16℃에서 150 rev/min로 교반하면서 배양하였다.For the protein expression of the enzyme, the transformed microorganisms stored frozen in Example 1 were aerated at 200 rev/min in a flask with 500 ml of LB (Difco, Sparks, MD, USA) medium and 20 μg/ml of kanamycin. Incubated at 37 ℃ under the conditions. When the absorbance of the bacteria reaches 0.6 to 0.8 at 600 nm, a final concentration of 0.1 mM IPTG is added to induce protein expression of the enzymes, and the culture is stirred for 16-18 hours at 16°C at 150 rev/min. cultured.

배양된 9-리폭시게나아제를 포함하는 대장균 세포를 모아서 사용하였다. 또한, 상기와 같이 과발현되어 생산된 9-리폭시게나아제는 상기 형질전환된 균주의 배양액을 6,000xg로 4℃에서 30분 동안 원심분리하여 0.85% 염화나트륨(NaCl)으로 두 번 세척한 다음 수산화지방산 또는 이수산화지방산을 생산하기 위한 재조합 세포로 사용하였다. 9-리폭시게나아제는 2단계 산화반응을 수행하여 이수산화지방산을 생산하므로, 위치 지정 돌연변이(site-dierected mutagenesis)를 통하여 1단계 산화반응을 잘 수행하는 9-리폭시게나아제 변이체(V569F)를 제조하여 수산화지방산 생산에 사용하였다.E. coli cells containing cultured 9-lipoxygenase were collected and used. In addition, the 9-lipoxygenase produced by overexpression as described above was centrifuged at 6,000xg at 4° C. for 30 minutes, washed twice with 0.85% sodium chloride (NaCl), and then hydroxylated fatty acid or It was used as a recombinant cell for producing fatty acid dihydrogen. Since 9-lipoxygenase produces dihydrogenated fatty acids by performing a two-step oxidation reaction, a 9-lipoxygenase variant (V569F) that performs well in a first-step oxidation reaction is prepared through site-directed mutagenesis. and used for the production of hydroxyl fatty acids.

실시예 3. 9-리폭시게나아제를 이용한 아라키돈산으로부터 9-수산화아라키돈산 및 9,15-이수산화아라키돈산의 합성경로 구축Example 3. Construction of synthesis route of 9-hydroxyarachidonic acid and 9,15-arachidonic acid dihydroxide from arachidonic acid using 9-lipoxygenase

상기 9-리폭시게나아제를 이용하여 9-수산화아라키돈산 및 9,15-이수산화아라키돈산의 합성경로를 구축하기 위하여 아라키돈산(ARA)을 기질로 사용하였다. 9-수산화아라키돈산 및 9,15-이수산화아라키돈산은 1 mM의 아라키돈산에 대하여, 9-리폭시게나아제 0.05 g/L를 사용하였으며 200 mM 시스테인을 첨가하고, pH 8.5 및 30℃에서 15 분 동안 수행하였다. Arachidonic acid (ARA) was used as a substrate to construct a synthesis pathway of 9-hydroxyarachidonic acid and 9,15-arachidonic acid dihydroxide using the 9-lipoxygenase. For 9-hydroxyarachidonic acid and 9,15-arachidonic acid dihydroxide, 0.05 g/L of 9-lipoxygenase was used for 1 mM of arachidonic acid, 200 mM cysteine was added, pH 8.5 and 30°C for 15 minutes. performed.

그 결과 도 1과 같은 합성경로를 구축하였다. 또한, 생산된 9-수산화아라키돈산 및 9,15-이수산화아라키돈산을 HPLC를 이용하여 확인하였으며 (도 2), 물질동정을 통해 9,15-이수산화아라키돈산을 확인하였다(도 3).As a result, a synthetic route as shown in FIG. 1 was constructed. In addition, the produced 9-arachidonic acid and 9,15-arachidonic acid dihydroxide were confirmed using HPLC (FIG. 2), and 9,15-arachidonic acid dihydroxide was confirmed through material identification (FIG. 3).

실시예 4. 9-리폭시게나아제를 이용한 에이코사펜타엔산으로부터 9-수산화에이코사펜타엔산 및 9,15-이수산화에이코사펜타엔산의 합성경로 구축Example 4. Construction of synthesis route of 9-hydroxyeicosapentaenoic acid and 9,15-dihydroxyeicosapentaenoic acid from eicosapentaenoic acid using 9-lipoxygenase

상기 9-리폭시게나아제를 이용하여 9-수산화에이코사펜타엔산 및 9,15-이수산화에이코사펜타엔산의 합성경로를 구축하기 위하여 에이코사펜타엔산 (EPA)을 기질로 사용하였다. 9-수산화에이코사펜타엔산 및 9,15-이수산화에이코사펜타엔산은 1 mM의 에이코사펜타엔산에 대하여, 9-리폭시게나아제 0.5 g/L를 사용하였으며 200 mM의 시스테인을 첨가하고 pH 8.5 및 30℃에서 60 분 동안 수행하였다.Eicosapentaenoic acid (EPA) was used as a substrate to construct a synthesis pathway for 9-hydroxyeicosapentaenoic acid and 9,15-dihydroxyeicosapentaenoic acid using the 9-lipoxygenase. For 9-hydroxyeicosapentaenoic acid and 9,15-dihydroxyeicosapentaenoic acid, 0.5 g/L of 9-lipoxygenase was used for 1 mM of eicosapentaenoic acid and 200 mM of cysteine was added, This was done at pH 8.5 and 30° C. for 60 minutes.

그 결과 도 4와 같은 합성경로를 구축하였다. 또한 생산된 9-수산화에이코사펜타엔산 및 9,15-이수산화에이코사펜타엔산을 HPLC를 이용하여 확인하였으며(도 5), 물질동정을 통해 9,15-이수산화에이코사펜타엔산을 확인하였다(도 6).As a result, a synthetic route as shown in FIG. 4 was constructed. In addition, the produced 9-hydroxyeicosapentaenoic acid and 9,15-eicosapentaenoic acid dihydroxide were confirmed using HPLC (FIG. 5), and 9,15-eicosapentaenoic acid dihydroxide was identified through material identification. was confirmed (FIG. 6).

실시예 5. 9-리폭시게나아제를 이용한 도코사펜타엔산으로부터 11-수산화도코사펜타엔산 및 11,17-이수산화도코사펜타엔산의 합성경로 구축Example 5. Construction of synthesis route of 11-hydroxydocosapentaenoic acid and 11,17-dihydroxydocosapentaenoic acid from docosapentaenoic acid using 9-lipoxygenase

상기 9-리폭시게나아제를 이용하여 11-수산화도코사펜타엔산 및 11,17-이수산화도코사펜타엔산의 합성경로를 구축하기 위하여 도코사펜타엔산(DPA)을 기질로 사용하였다. 11-수산화도코사펜타엔산 및 11,17-이수산화도코사펜타엔산은 1 mM의 도코사펜타엔산에 대하여, 9-리폭시게나아제 0.1 g/L를 사용하였으며 200 mM의 시스테인을 첨가하고 pH 8.5 및 30℃에서 20 분 동안 수행하였다.Docosapentaenoic acid (DPA) was used as a substrate to construct a synthesis pathway for 11-hydroxydocosapentaenoic acid and 11,17-dihydroxydocosapentaenoic acid using the 9-lipoxygenase. For 11-hydroxydocosapentaenoic acid and 11,17-dihydroxydocosapentaenoic acid, 0.1 g/L of 9-lipoxygenase was used for 1 mM of docosapentaenoic acid, 200 mM of cysteine was added, and pH 8.5 and 30 °C for 20 minutes.

그 결과 도 7과 같은 합성경로를 구축하였다. 또한 생산된 11-수산화도코사펜타엔산 및 11,17-이수산화도코사펜타엔산을 HPLC를 이용하여 확인하였으며(도 8), 물질동정을 통해 11,17-이수산화도코사펜타엔산을 확인하였다(도 9).As a result, a synthetic route as shown in FIG. 7 was constructed. In addition, the produced 11-hydroxydocosapentaenoic acid and 11,17-dihydroxydocosapentaenoic acid were confirmed using HPLC (FIG. 8), and 11,17-dihydroxydocosapentaenoic acid was confirmed through material identification. (FIG. 9).

실시예 6. 9-리폭시게나아제를 이용한 도코사헥사엔산으로부터 11-수산화도코사헥사엔산 및 11,17-이수산화도코사헥사엔산의 합성경로 구축Example 6. Construction of synthesis route of 11-hydroxydocosahexaenoic acid and 11,17-dihydroxide docosahexaenoic acid from docosahexaenoic acid using 9-lipoxygenase

상기 9-리폭시게나아제를 이용하여 11-수산화도코사헥사엔산 및 11,17-이수산화도코사헥사엔산의 합성경로를 구축하기 위하여 도코사헥사엔산(DHA)을 기질로 사용하였다. 11-수산화도코사헥사엔산 및 11,17-이수산화도코사헥사엔산은 1 mM의 도코사헥사엔산에 대하여, 9-리폭시게나아제 0.1 g/L를 사용하였으며 200 mM의 시스테인을 첨가하고 pH 8.5 및 30℃에서 20 분 동안 수행하였다.Docosahexaenoic acid (DHA) was used as a substrate in order to construct a synthesis pathway for 11-hydroxydocosahexaenoic acid and 11,17-dihydroxydocosahexaenoic acid using the 9-lipoxygenase. For 11-hydroxydocosahexaenoic acid and 11,17-dihydrodocosahexaenoic acid, 0.1 g/L of 9-lipoxygenase was used for 1 mM of docosahexaenoic acid, 200 mM of cysteine was added, and pH 8.5 and 30 °C for 20 minutes.

그 결과 도 10과 같은 합성경로를 구축하였다. 또한 생산된 11-수산화도코사헥사엔산과 11,17-이수산화도코사헥사엔산을 HPLC를 이용하여 확인하였으며(도 11), 물질동정을 통해 11,17-이수산화도코사헥사엔산을 확인하였다(도 12).As a result, a synthetic route as shown in FIG. 10 was constructed. In addition, the produced 11-hydroxydocosahexaenoic acid and 11,17-dihydrogenated docosahexaenoic acid were confirmed using HPLC (FIG. 11), and 11,17-dihydrogenated docosahexaenoic acid was confirmed through material identification. (FIG. 12).

실시예 7. 9-리폭시게나아제의 pH 및 온도가 9-수산화아라키돈산, 9,15-이수산화아라키돈산의 생산 활성에 미치는 영향Example 7. Effect of pH and temperature of 9-lipoxygenase on production activity of 9-hydroxyarachidonic acid and 9,15-arachidonic acid dihydroxide

상기 9-리폭시게나아제의 9-수산화아라키돈산 및 9,15-이수산화아라키돈산의 생산에 대한 pH 효과를 조사하기 위하여, 기질로서 1 mM의 아라키돈산에 대하여 헤페스 완충용액(HEPES buffer, pH 6.5-7.5), 이피피에스 완충용액(EPPS buffer, pH 7.5-8.5) 및 체스 완충용액(CHES buffer, pH 8.5-9.0)에서 10분 동안 효소반응을 수행하였다. 그 결과, 도 13a에 나타난 바와 같이, 최적 pH는 8.5인 것을 알 수 있었다.In order to investigate the pH effect of the 9-lipoxygenase on the production of 9-hydroxyarachidonic acid and 9,15-arachidonic acid dihydroxide, Hepes buffer solution (HEPES buffer, pH 6.5-7.5), EPPS buffer (EPPS buffer, pH 7.5-8.5) and Ches buffer (CHES buffer, pH 8.5-9.0) for 10 minutes. As a result, as shown in Figure 13a, it was found that the optimum pH was 8.5.

또한, 상기 9-리폭시게나아제의 9-수산화아라키돈산 및 9,15-이수산화아라키돈산의 생산에 대한 온도의 효과를 조사하기 위하여, 기질로서 1 mM의 아라키돈산에 대하여 50 mM EPPS 완충용액 pH 8.5에서, 온도를 20℃에서 40℃까지 5℃ 간격으로 범위를 정한 뒤, 10분 동안 효소 반응을 수행하였다. 그 결과, 도 13b에 나타난 바와 같이, 최적 온도는 30℃인 것을 알 수 있었다.In addition, in order to investigate the effect of temperature on the production of 9-hydroxarachidonic acid and 9,15-arachidonic acid dihydroxide by the 9-lipoxygenase, 50 mM EPPS buffer solution pH with respect to 1 mM arachidonic acid as a substrate At 8.5, the temperature was ranged from 20 °C to 40 °C at 5 °C intervals, followed by an enzymatic reaction for 10 min. As a result, as shown in FIG. 13B, it was found that the optimum temperature was 30°C.

실시예 8. 9-리폭시게나아제 변이체(V569F)를 이용한 9-수산화아라키돈산의 전환률Example 8. Conversion rate of 9-hydroxyarachidonic acid using 9-lipoxygenase variant (V569F)

본 발명의 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) 유래 9-리폭시게나아제 변이체(V569F)를 이용한 9-수산화아라키돈산의 생산을 확인하기 위하여, 9-리폭시게나아제 변이체(V569F) 0.05 g/L 를 함유한 50 mM EPPS 완충용액을 pH 8.5 및 온도 30℃에서 1 mM의 아라키돈산을 기질로 하여 9-수산화아라키돈산의 시간별 전환률을 측정하였다. 그 결과를 도 14에 나타내었고, 9-리폭시게나아제 변이체(V569F)는 0.95 mM의 9-수산화아라키돈산을생산하였다 최종 9-수산화아라키돈산의 전환 수율은 95 %로 나타났다.In order to confirm the production of 9-hydroxyarachidonic acid using the 9-lipoxygenase variant (V569F) derived from Sphingomonas macrogoltabidus of the present invention, the 9-lipoxygenase variant (V569F) 0.05 g / The conversion rate of 9-hydroxyarachidonic acid over time was measured using 1 mM arachidonic acid as a substrate in a 50 mM EPPS buffer solution containing L at pH 8.5 and a temperature of 30 ° C. The results are shown in FIG. 14, and the 9-lipoxygenase variant (V569F) produced 0.95 mM of 9-hydroxyarachidonic acid. The final conversion yield of 9-hydroxyarachidonic acid was 95%.

실시예 9. 9-리폭시게나아제를 이용한 9,15-이수산화아라키돈산의 전환률Example 9. Conversion rate of 9,15-arachidonic acid dihydroxide using 9-lipoxygenase

본 발명의 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) 유래 9-리폭시게나아제를 이용한 9,15-이수산화아라키돈산의 생산을 확인하기 위하여, 9-리폭시게나아제 0.05 g/L 를 함유한 50 mM EPPS 완충용액을 pH 8.5 및 온도 30℃에서 1 mM의 아라키돈산을 기질로 하여 9,15-이수산화아라키돈산의 시간별 전환률을 측정하였다. 그 결과를 도 15에 나타내었고, 9-리폭시게나아제는 1 mM의 9,15-이수산화아라키돈산을 생산하였다 최종 9,15-이수산화아라키돈산의 전환 수율은 100 %로 나타났다.In order to confirm the production of 9,15-arachidonic acid dihydroxide using 9-lipoxygenase derived from Sphingomonas macrogoltabidus of the present invention, 50 g/L containing 0.05 g/L of 9-lipoxygenase The conversion rate of 9,15-arachidonic acid dihydroxide over time was measured using 1 mM arachidonic acid as a substrate in mM EPPS buffer solution at pH 8.5 and temperature 30°C. The results are shown in FIG. 15, and 9-lipoxygenase produced 1 mM of 9,15-arachidonic acid dihydroxide. The conversion yield of the final 9,15-arachidonic acid dihydroxide was 100%.

실시예 10. 9-리폭시게나아제 변이체(V569F)를 이용한 9-수산화에이코사펜타엔산의 전환률Example 10. Conversion rate of 9-hydroxyeicosapentaenoic acid using a 9-lipoxygenase variant (V569F)

본 발명의 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) 유래 9-리폭시게나아제 변이체(V569F)를 이용한 9-수산화에이코사펜타엔산의 생산을 확인하기 위하여, 9-리폭시게나아제 변이체(V569F) 0.1 g/L 를 함유한 50 mM EPPS 완충용액을 pH 8.5 및 온도 30℃에서 1 mM의 에이코사펜타엔산을 기질로 하여 9-수산화에이코사펜타엔산의 시간별 전환률을 측정하였다. 그 결과를 도 16에 나타내었고, 9-리폭시게나아제 변이체(V569F)는 0.72 mM의 9-수산화에이코사펜타엔산을 생산하였다 최종 9-수산화에이코사펜타엔산의 전환 수율은 72 %로 나타났다.In order to confirm the production of 9-hydroxyeicosapentaenoic acid using the 9-lipoxygenase variant (V569F) derived from Sphingomonas macrogoltabidus of the present invention, the 9-lipoxygenase variant (V569F) The conversion rate of 9-hydroxyeicosapentaenoic acid over time was measured using 1 mM eicosapentaenoic acid as a substrate in a 50 mM EPPS buffer solution containing 0.1 g/L at pH 8.5 and a temperature of 30° C. The results are shown in FIG. 16, and the 9-lipoxygenase variant (V569F) produced 0.72 mM of 9-hydroxyeicosapentaenoic acid. The final conversion yield of 9-hydroxyeicosapentaenoic acid was 72%. .

실시예 11. 9-리폭시게나아제를 이용한 9,15-이수산화에이코사펜타엔산의 전환률Example 11. Conversion rate of 9,15-dihydroxyeicosapentaenoic acid using 9-lipoxygenase

본 발명의 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) 유래 9-리폭시게나아제를 이용한 9,15-이수산화에이코사펜타엔산의 생산을 확인하기 위하여, 9-리폭시게나아제 0.5 g/L를 함유한 50 mM EPPS 완충용액을 pH 8.5 및 온도 30℃에서 1 mM의 에이코사펜타엔산을 기질로 하여 9,15-이수산화에이코사펜타엔산의 시간별 전환률을 측정하였다. 그 결과를 도 17에 나타내었고, 9-리폭시게나아제는 0.83 mM의 9,15-이수산화에이코사펜타엔산을 생산하였다. 최종 9,15-이수산화에이코사펜타엔산의 전환 수율은 83 %로 나타났다.In order to confirm the production of 9,15-dihydroxyeicosapentaenoic acid using 9-lipoxygenase derived from Sphingomonas macrogoltabidus of the present invention, 0.5 g/L of 9-lipoxygenase The conversion rate of 9,15-dihydroxyeicosapentaenoic acid over time was measured using 1 mM eicosapentaenoic acid as a substrate in a 50 mM EPPS buffer solution containing 50 mM EPPS at pH 8.5 and a temperature of 30° C. The results are shown in FIG. 17, and 9-lipoxygenase produced 0.83 mM of 9,15-dihydroxyeicosapentaenoic acid. The final conversion yield of 9,15-dihydroxyeicosapentaenoic acid was 83%.

실시예 12. 9-리폭시게나아제 변이체(V569F)를 이용한 11-수산화도코사펜타엔산의 전환률Example 12. Conversion rate of 11-hydroxydocosapentaenoic acid using 9-lipoxygenase variant (V569F)

본 발명의 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) 유래 9-리폭시게나아제 변이체(V569F)를 이용한 11-수산화도코사펜타엔산의 생산을 확인하기 위하여, 9-리폭시게나아제 변이체(V569F) 0.1 g/L 를 함유한 50 mM EPPS 완충용액을 pH 8.5 및 온도 30℃에서 1 mM의 도코사펜타엔산을 기질로 하여 11-수산화도코사펜타엔산의 시간별 전환률을 측정하였다. 그 결과를 도 18에 나타내었고, 9-리폭시게나아제 변이체(V569F)는 0.76 mM의 11-수산화도코사펜타엔산을 생산하였다 최종 11-수산화도코사펜타엔산의 전환 수율은 76 %로 나타났다.In order to confirm the production of 11-hydroxydocosapentaenoic acid using the 9-lipoxygenase mutant (V569F) derived from Sphingomonas macrogoltabidus of the present invention, the 9-lipoxygenase mutant (V569F) The conversion rate of 11-hydroxydocosapentaenoic acid over time was measured using 1 mM docosapentaenoic acid as a substrate in a 50 mM EPPS buffer solution containing 0.1 g/L at pH 8.5 and a temperature of 30°C. The results are shown in FIG. 18, and the 9-lipoxygenase variant (V569F) produced 0.76 mM of 11-hydroxydocosapentaenoic acid. The final conversion yield of 11-hydroxydocosapentaenoic acid was 76%. .

실시예 13. 9-리폭시게나아제를 이용한 11,17-이수산화도코사펜타엔산의 전환률Example 13. Conversion rate of 11,17-dihydroxydocosapentaenoic acid using 9-lipoxygenase

본 발명의 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) 유래 9-리폭시게나아제를 이용한 11,17-이수산화도코사펜타엔산의 생산을 확인하기 위하여, 9-리폭시게나아제 0.1 g/L 를 함유한 50 mM EPPS 완충용액을 pH 8.5 및 온도 30℃에서 1 mM의 도코사펜타엔산을 기질로 하여 11,17-이수산화도코사펜타엔산의 시간별 전환률을 측정하였다. 그 결과를 도 19에 나타내었고, 9-리폭시게나아제는 0.94 mM의 11,17-이수산화도코사펜타엔산을 생산하였다. 최종 11,17-이수산화도코사펜타엔산의 전환 수율은 94 %로 나타났다.In order to confirm the production of 11,17-dihydroxydocosapentaenoic acid using 9-lipoxygenase derived from Sphingomonas macrogoltabidus of the present invention, 0.1 g/L of 9-lipoxygenase was contained. The conversion rate of 11,17-dihydrodocosapentaenoic acid over time was measured using 1 mM docosapentaenoic acid as a substrate in a 50 mM EPPS buffer at pH 8.5 and at a temperature of 30° C. The results are shown in FIG. 19, and 9-lipoxygenase produced 0.94 mM of 11,17-dihydroxydocosapentaenoic acid. The final conversion yield of 11,17-dihydroxydocosapentaenoic acid was 94%.

실시예 14. 9-리폭시게나아제 변이체(V569F)를 이용한 11-수산화도코사헥사엔산의 전환률Example 14. Conversion rate of 11-hydroxydocosahexaenoic acid using 9-lipoxygenase variant (V569F)

본 발명의 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) 유래 9-리폭시게나아제 변이체(V569F)를 이용한 11-수산화도코사헥사엔산의 생산을 확인하기 위하여, 9-리폭시게나아제 변이체(V569F) 0.1 g/L 를 함유한 50 mM EPPS 완충용액을 pH 8.5 및 온도 30℃에서 1 mM의 도코사헥사엔산을 기질로 하여 11-수산화도코사헥사엔산의 시간별 전환률을 측정하였다. 그 결과를 도 20에 나타내었고, 9-리폭시게나아제 변이체(V569F)는 0.78 mM의 11-수산화도코사헥사엔산을 생산하였다 최종 11-수산화도코사헥사엔산의 전환 수율은 78 %로 나타났다.In order to confirm the production of 11-hydroxydocosahexaenoic acid using the 9-lipoxygenase mutant (V569F) derived from Sphingomonas macrogoltabidus of the present invention, the 9-lipoxygenase mutant (V569F) The conversion rate of 11-hydroxydocosahexaenoic acid over time was measured using 1 mM docosahexaenoic acid as a substrate in a 50 mM EPPS buffer solution containing 0.1 g/L at pH 8.5 and a temperature of 30° C. The results are shown in FIG. 20, and the 9-lipoxygenase variant (V569F) produced 0.78 mM of 11-hydroxydocosahexaenoic acid. The final conversion yield of 11-hydroxydocosahexaenoic acid was 78%. .

실시예 15. 9-리폭시게나아제를 이용한 11,17-이수산화도코사헥사엔산의 전환률Example 15. Conversion rate of 11,17-dihydroxide docosahexaenoic acid using 9-lipoxygenase

본 발명의 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) 유래 9-리폭시게나아제를 이용한 11,17-이수산화도코사헥사엔산의 생산을 확인하기 위하여, 9-리폭시게나아제 0.1 g/L 를 함유한 50 mM EPPS 완충용액을 pH 8.5 및 온도 30℃에서 1 mM의 도코사헥사엔산을 기질로 하여 11,17-이수산화도코사헥사엔산의 시간별 전환률을 측정하였다. 그 결과를 도 21에 나타내었고, 9-리폭시게나아제는 0.95 mM의 11,17-이수산화도코사헥사엔산을 생산하였다. 최종 11,17-이수산화도코사헥사엔산의 전환 수율은 95 %로 나타났다.In order to confirm the production of 11,17-dihydroxydocosahexaenoic acid using 9-lipoxygenase derived from Sphingomonas macrogoltabidus of the present invention, 0.1 g/L of 9-lipoxygenase was contained. The conversion rate of 11,17-dihydroxide docosahexaenoic acid over time was measured using 1 mM docosahexaenoic acid as a substrate in a 50 mM EPPS buffer at pH 8.5 and at a temperature of 30° C. The results are shown in FIG. 21, and 9-lipoxygenase produced 0.95 mM of 11,17-dihydroxydocosahexaenoic acid. The final conversion yield of 11,17-dihydroxydocosahexaenoic acid was 95%.

이상, 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 지방산업계 및 의료계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구 항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.In the above, a specific part of the content of the present invention has been described in detail, for those skilled in the fatty acid industry and the medical field, these specific techniques are only preferred embodiments, thereby not limiting the scope of the present invention. The point will be clear. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Konkuk University Industrial Cooperation Corp <120> COMPOSITION AND METHOD FOR PRODUCING HYDROXY FATTY ACID OR DIHYDROXY FATTY ACID USING 9-LIPOXYGENASE OR VARIANT THEREOF <130> 2020-I141 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 646 <212> PRT <213> Sphingomonas macrogoltabidus <400> 1 Met Pro Ser Ala Asn Pro Ser Leu Pro Gln Asn Asp Thr Pro Ala Glu 1 5 10 15 Gln Ala Ala Arg Ala Ala Gln Leu Ala Ala Ser Gln Ala Val Tyr Val 20 25 30 Trp Thr Thr Asp Val Pro Thr Leu Pro Gly Val Pro Leu Ala Thr Asp 35 40 45 Val Pro Lys Asn Asp Glu Pro Thr Ile Ala Trp Phe Gly Ile Leu Ile 50 55 60 Gly Val Gly Leu Ala Ile Val Arg Asn Ala Leu Thr Val Lys Leu Gly 65 70 75 80 Gly Val Asp Arg Gly Glu Leu Asp Thr Pro Arg Ala Glu Tyr Glu Thr 85 90 95 Ala Leu Ala Glu Cys Asp Ala Ile Glu Leu Ser Thr Ala Lys Ile Val 100 105 110 Ala Glu His Gly Val His Ser Gly Gly Asn Ile Phe Glu Arg Ile Val 115 120 125 Gly Asp Val Glu Asn Ala Val Ala Ala Ala Glu Arg Asp Val His Leu 130 135 140 Ala Leu Leu Gln Gly Tyr Lys Glu Arg Leu Glu Asp Leu Met Lys Val 145 150 155 160 Asp Glu Ala Glu Val Ala Gly Leu Gly Ser Lys Thr Pro Arg Ser Leu 165 170 175 Asp Ala Tyr Arg Ala Leu Phe Ala Thr Leu Pro Val Pro Gly Ile Ser 180 185 190 Tyr Met Phe Gln Asp Asp Lys Glu Phe Ala Arg Leu Arg Val Gln Gly 195 200 205 Pro Asn Cys Met Leu Ile Ala Ala Val Glu Gly Ala Leu Pro Ala Asn 210 215 220 Phe Pro Leu Ser Glu Lys Ala Tyr Ala Ala Val Val Asn Gly Asp Thr 225 230 235 240 Leu Ala Ala Ala Leu Ala Asp Gly Arg Leu Phe Met Leu Asp Tyr Lys 245 250 255 Pro Leu Ala Ile Leu Asp Pro Gly Thr Tyr Gly Gly Glu Ala Lys Tyr 260 265 270 Val Ser Gln Pro Met Ala Leu Phe Ala Val Pro Pro Gly Gly Ala Ser 275 280 285 Leu Ile Pro Val Ala Ile Gln Cys Gly Gln Asp Pro Ala Asp Cys Pro 290 295 300 Ile Phe Thr Pro Ser Pro Ala Ala Asp Arg Gln Trp Gly Trp Glu Met 305 310 315 320 Ala Lys Phe Val Val Gln Val Ala Asp Gly Asn Tyr His Glu Leu Phe 325 330 335 Ala His Leu Ala Arg Thr His Leu Val Ile Glu Ala Phe Ala Val Ala 340 345 350 Thr His Arg His Leu Ala Glu Ala His Pro Val Trp Ala Leu Leu Val 355 360 365 Pro His Phe Glu Gly Thr Leu Phe Ile Asn Glu Gln Ala Ala Thr Ser 370 375 380 Leu Ile Ala Ala Asn Gly Pro Ile Asp His Ile Phe Ala Gly Thr Ile 385 390 395 400 Ala Ser Ser Gln Leu Ala Ala Val Asp Ala Arg Leu Ala Phe Asp Phe 405 410 415 Arg Gly Lys Met Pro His Ala Asp Phe Ala Ala Arg Gly Val Gly Val 420 425 430 Asp Ser Ala Leu Ala Asp Tyr Pro Tyr Arg Asp Asp Ala Leu Leu Val 435 440 445 Trp Asp Ala Ile His Glu Trp Ala Arg Gln Tyr Val Asp Leu Tyr Tyr 450 455 460 Thr Gly Asp Thr Asp Ile Val Ala Asp Thr Glu Leu Ala Ala Trp Ala 465 470 475 480 Ala Cys Leu Ala Gly Glu Ala Lys Val Gly Gly Leu Gly Pro Val Thr 485 490 495 Thr Arg Asn Gln Leu Ala Glu Ile Cys Ala Met Val Met Phe Thr Ala 500 505 510 Ser Ala Gln His Ala Ala Val Asn Leu Pro Gln Lys Asp Ile Met Ala 515 520 525 Phe Ala Pro Ala Val Thr Gly Ala Ala Trp Gln Pro Ala Pro Asn Gly 530 535 540 Gln Arg Gly His Asp Lys Thr Gly Trp Leu Ala Met Met Pro Pro Met 545 550 555 560 Ala Leu Ala Leu Glu Gln Leu Asn Val Leu Glu Leu Leu Gly Ser Val 565 570 575 His Tyr Arg Pro Leu Gly Asp Tyr Arg Ser Asn Ala Phe Pro Tyr Pro 580 585 590 Gln Trp Phe Gln Asp Pro Arg Val Thr Ala Ala Glu Gly Pro Leu Ala 595 600 605 Trp Phe Gln Ala Ala Leu Ala Asp Val Glu Ala Glu Ile Val Thr Arg 610 615 620 Asn Ala Glu Arg Met Gln Pro Tyr Pro Tyr Leu Gln Pro Ser Leu Ile 625 630 635 640 Pro Thr Ser Ile Asn Ile 645 <210> 2 <211> 646 <212> PRT <213> Artificial Sequence <220> <223> V569F mutant of 9-lipoxygenase <400> 2 Met Pro Ser Ala Asn Pro Ser Leu Pro Gln Asn Asp Thr Pro Ala Glu 1 5 10 15 Gln Ala Ala Arg Ala Ala Gln Leu Ala Ala Ser Gln Ala Val Tyr Val 20 25 30 Trp Thr Thr Asp Val Pro Thr Leu Pro Gly Val Pro Leu Ala Thr Asp 35 40 45 Val Pro Lys Asn Asp Glu Pro Thr Ile Ala Trp Phe Gly Ile Leu Ile 50 55 60 Gly Val Gly Leu Ala Ile Val Arg Asn Ala Leu Thr Val Lys Leu Gly 65 70 75 80 Gly Val Asp Arg Gly Glu Leu Asp Thr Pro Arg Ala Glu Tyr Glu Thr 85 90 95 Ala Leu Ala Glu Cys Asp Ala Ile Glu Leu Ser Thr Ala Lys Ile Val 100 105 110 Ala Glu His Gly Val His Ser Gly Gly Asn Ile Phe Glu Arg Ile Val 115 120 125 Gly Asp Val Glu Asn Ala Val Ala Ala Ala Glu Arg Asp Val His Leu 130 135 140 Ala Leu Leu Gln Gly Tyr Lys Glu Arg Leu Glu Asp Leu Met Lys Val 145 150 155 160 Asp Glu Ala Glu Val Ala Gly Leu Gly Ser Lys Thr Pro Arg Ser Leu 165 170 175 Asp Ala Tyr Arg Ala Leu Phe Ala Thr Leu Pro Val Pro Gly Ile Ser 180 185 190 Tyr Met Phe Gln Asp Asp Lys Glu Phe Ala Arg Leu Arg Val Gln Gly 195 200 205 Pro Asn Cys Met Leu Ile Ala Ala Val Glu Gly Ala Leu Pro Ala Asn 210 215 220 Phe Pro Leu Ser Glu Lys Ala Tyr Ala Ala Val Val Asn Gly Asp Thr 225 230 235 240 Leu Ala Ala Ala Leu Ala Asp Gly Arg Leu Phe Met Leu Asp Tyr Lys 245 250 255 Pro Leu Ala Ile Leu Asp Pro Gly Thr Tyr Gly Gly Glu Ala Lys Tyr 260 265 270 Val Ser Gln Pro Met Ala Leu Phe Ala Val Pro Pro Gly Gly Ala Ser 275 280 285 Leu Ile Pro Val Ala Ile Gln Cys Gly Gln Asp Pro Ala Asp Cys Pro 290 295 300 Ile Phe Thr Pro Ser Pro Ala Ala Asp Arg Gln Trp Gly Trp Glu Met 305 310 315 320 Ala Lys Phe Val Val Gln Val Ala Asp Gly Asn Tyr His Glu Leu Phe 325 330 335 Ala His Leu Ala Arg Thr His Leu Val Ile Glu Ala Phe Ala Val Ala 340 345 350 Thr His Arg His Leu Ala Glu Ala His Pro Val Trp Ala Leu Leu Val 355 360 365 Pro His Phe Glu Gly Thr Leu Phe Ile Asn Glu Gln Ala Ala Thr Ser 370 375 380 Leu Ile Ala Ala Asn Gly Pro Ile Asp His Ile Phe Ala Gly Thr Ile 385 390 395 400 Ala Ser Ser Gln Leu Ala Ala Val Asp Ala Arg Leu Ala Phe Asp Phe 405 410 415 Arg Gly Lys Met Pro His Ala Asp Phe Ala Ala Arg Gly Val Gly Val 420 425 430 Asp Ser Ala Leu Ala Asp Tyr Pro Tyr Arg Asp Asp Ala Leu Leu Val 435 440 445 Trp Asp Ala Ile His Glu Trp Ala Arg Gln Tyr Val Asp Leu Tyr Tyr 450 455 460 Thr Gly Asp Thr Asp Ile Val Ala Asp Thr Glu Leu Ala Ala Trp Ala 465 470 475 480 Ala Cys Leu Ala Gly Glu Ala Lys Val Gly Gly Leu Gly Pro Val Thr 485 490 495 Thr Arg Asn Gln Leu Ala Glu Ile Cys Ala Met Val Met Phe Thr Ala 500 505 510 Ser Ala Gln His Ala Ala Val Asn Leu Pro Gln Lys Asp Ile Met Ala 515 520 525 Phe Ala Pro Ala Val Thr Gly Ala Ala Trp Gln Pro Ala Pro Asn Gly 530 535 540 Gln Arg Gly His Asp Lys Thr Gly Trp Leu Ala Met Met Pro Pro Met 545 550 555 560 Ala Leu Ala Leu Glu Gln Leu Asn Phe Leu Glu Leu Leu Gly Ser Val 565 570 575 His Tyr Arg Pro Leu Gly Asp Tyr Arg Ser Asn Ala Phe Pro Tyr Pro 580 585 590 Gln Trp Phe Gln Asp Pro Arg Val Thr Ala Ala Glu Gly Pro Leu Ala 595 600 605 Trp Phe Gln Ala Ala Leu Ala Asp Val Glu Ala Glu Ile Val Thr Arg 610 615 620 Asn Ala Glu Arg Met Gln Pro Tyr Pro Tyr Leu Gln Pro Ser Leu Ile 625 630 635 640 Pro Thr Ser Ile Asn Ile 645 <210> 3 <211> 1941 <212> DNA <213> Sphingomonas macrogoltabidus <400> 3 ttgccatcgg ccaatcccag cctgccgcaa aacgatacgc ccgccgagca agcggcgcgc 60 gcggcacagc tcgccgcttc gcaggccgtc tatgtctgga ccaccgacgt cccgacgctg 120 cccggcgtgc cgctcgccac cgacgtgccg aagaacgacg aaccgacgat cgcctggttc 180 ggcatcttga tcggcgtcgg cctcgcgatc gtgcgcaatg cgctgacggt gaagctcggc 240 ggcgtcgacc ggggcgagct cgacacgccg cgcgccgaat atgaaaccgc gctcgccgag 300 tgcgacgcga tcgagctatc gaccgcgaag atcgtcgccg aacatggcgt tcacagcggc 360 ggcaatatct tcgaacgcat cgtcggcgac gtggaaaatg ccgtcgccgc ggctgagcgc 420 gacgtgcatc tcgctttgct tcagggatat aaggagcggc tcgaggatct catgaaggtc 480 gacgaggccg aggtggcggg gctgggcagc aagacgccgc gcagcctcga cgcatatcgc 540 gcgctgttcg ctaccctgcc ggtgccgggc atcagctata tgttccagga cgacaaggag 600 ttcgcccgcc tgcgcgtgca ggggccgaac tgcatgctga tcgcggcggt cgagggagcg 660 ctgcccgcca atttcccgct gagcgaaaag gcttacgccg cggtggtgaa tggcgacacg 720 ctggccgccg cgctcgccga cggccgcctc ttcatgctcg attacaagcc gctcgcgatc 780 ctcgaccccg gcacctatgg cggcgaagcc aaatatgtct cgcagccgat ggcgctgttc 840 gcggtaccgc cgggcggcgc atcgctcatc cccgtcgcga tccagtgcgg ccaggacccc 900 gccgactgtc cgattttcac gccctcaccg gcggccgaca ggcaatgggg ctgggaaatg 960 gcgaaattcg tcgtgcaggt cgccgacggt aattatcatg agctcttcgc ccatctggca 1020 cgcacccatc tggtgatcga ggcgttcgcg gtcgcgacgc accgccatct tgccgaggcg 1080 catccggtgt gggcgctgct cgtcccgcat ttcgagggga cattgttcat caacgaacag 1140 gcggcgacct cgctgatcgc ggcgaacggc ccgatcgacc atattttcgc ggggacgatc 1200 gcgtcgagcc agcttgccgc agtcgatgcg cggctggcgt tcgatttccg gggcaagatg 1260 ccgcacgccg attttgccgc gcggggcgtc ggggtcgatt cggcgctcgc cgactatccg 1320 tatcgcgacg acgcgctgct ggtgtgggac gcgatccacg aatgggcgcg gcaatatgtc 1380 gatctttatt atacgggcga caccgatatc gtcgccgata ccgagctggc ggcatgggcc 1440 gcgtgcctcg cgggcgaagc caaggtcggc gggctcggcc cggtgacgac gcgcaaccag 1500 ctcgccgaaa tctgcgcgat ggtgatgttc accgccagcg cgcagcatgc ggcggtcaat 1560 ttaccgcaaa aggacatcat ggccttcgcc cccgcggtga ccggcgcagc gtggcaaccg 1620 gcgccgaacg gccagcgcgg acacgacaag acgggctggc tcgcgatgat gccgccgatg 1680 gccctcgcgc tcgaacaatt gaacgtgctc gaactgctgg ggtcggtgca ttatcgcccg 1740 ctcggcgact atcgcagcaa cgcctttccc tatccgcagt ggttccagga tccgcgcgtc 1800 accgcggcgg agggcccgct cgcctggttt caggcggcgc ttgccgacgt cgaggcggaa 1860 atcgtcacgc gcaatgccga gcggatgcag ccctatccct atctgcagcc gagcctgatc 1920 ccgacgagca tcaatatctg a 1941 <210> 4 <211> 1941 <212> DNA <213> Artificial Sequence <220> <223> V569F mutant of 9-lipoxygenase <400> 4 ttgccatcgg ccaatcccag cctgccgcaa aacgatacgc ccgccgagca agcggcgcgc 60 gcggcacagc tcgccgcttc gcaggccgtc tatgtctgga ccaccgacgt cccgacgctg 120 cccggcgtgc cgctcgccac cgacgtgccg aagaacgacg aaccgacgat cgcctggttc 180 ggcatcttga tcggcgtcgg cctcgcgatc gtgcgcaatg cgctgacggt gaagctcggc 240 ggcgtcgacc ggggcgagct cgacacgccg cgcgccgaat atgaaaccgc gctcgccgag 300 tgcgacgcga tcgagctatc gaccgcgaag atcgtcgccg aacatggcgt tcacagcggc 360 ggcaatatct tcgaacgcat cgtcggcgac gtggaaaatg ccgtcgccgc ggctgagcgc 420 gacgtgcatc tcgctttgct tcagggatat aaggagcggc tcgaggatct catgaaggtc 480 gacgaggccg aggtggcggg gctgggcagc aagacgccgc gcagcctcga cgcatatcgc 540 gcgctgttcg ctaccctgcc ggtgccgggc atcagctata tgttccagga cgacaaggag 600 ttcgcccgcc tgcgcgtgca ggggccgaac tgcatgctga tcgcggcggt cgagggagcg 660 ctgcccgcca atttcccgct gagcgaaaag gcttacgccg cggtggtgaa tggcgacacg 720 ctggccgccg cgctcgccga cggccgcctc ttcatgctcg attacaagcc gctcgcgatc 780 ctcgaccccg gcacctatgg cggcgaagcc aaatatgtct cgcagccgat ggcgctgttc 840 gcggtaccgc cgggcggcgc atcgctcatc cccgtcgcga tccagtgcgg ccaggacccc 900 gccgactgtc cgattttcac gccctcaccg gcggccgaca ggcaatgggg ctgggaaatg 960 gcgaaattcg tcgtgcaggt cgccgacggt aattatcatg agctcttcgc ccatctggca 1020 cgcacccatc tggtgatcga ggcgttcgcg gtcgcgacgc accgccatct tgccgaggcg 1080 catccggtgt gggcgctgct cgtcccgcat ttcgagggga cattgttcat caacgaacag 1140 gcggcgacct cgctgatcgc ggcgaacggc ccgatcgacc atattttcgc ggggacgatc 1200 gcgtcgagcc agcttgccgc agtcgatgcg cggctggcgt tcgatttccg gggcaagatg 1260 ccgcacgccg attttgccgc gcggggcgtc ggggtcgatt cggcgctcgc cgactatccg 1320 tatcgcgacg acgcgctgct ggtgtgggac gcgatccacg aatgggcgcg gcaatatgtc 1380 gatctttatt atacgggcga caccgatatc gtcgccgata ccgagctggc ggcatgggcc 1440 gcgtgcctcg cgggcgaagc caaggtcggc gggctcggcc cggtgacgac gcgcaaccag 1500 ctcgccgaaa tctgcgcgat ggtgatgttc accgccagcg cgcagcatgc ggcggtcaat 1560 ttaccgcaaa aggacatcat ggccttcgcc cccgcggtga ccggcgcagc gtggcaaccg 1620 gcgccgaacg gccagcgcgg acacgacaag acgggctggc tcgcgatgat gccgccgatg 1680 gccctcgcgc tcgaacaatt gaacttcctc gaactgctgg ggtcggtgca ttatcgcccg 1740 ctcggcgact atcgcagcaa cgcctttccc tatccgcagt ggttccagga tccgcgcgtc 1800 accgcggcgg agggcccgct cgcctggttt caggcggcgc ttgccgacgt cgaggcggaa 1860 atcgtcacgc gcaatgccga gcggatgcag ccctatccct atctgcagcc gagcctgatc 1920 ccgacgagca tcaatatctg a 1941 <110> Konkuk University Industrial Cooperation Corp. <120> COMPOSITION AND METHOD FOR PRODUCING HYDROXY FATTY ACID OR DIHYDROXY FATTY ACID USING 9-LIPOXYGENASE OR VARIANT THEREOF <130> 2020-I141 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 646 <212> PRT <213> Sphingomonas macrogoltabidus <400> 1 Met Pro Ser Ala Asn Pro Ser Leu Pro Gln Asn Asp Thr Pro Ala Glu 1 5 10 15 Gln Ala Ala Arg Ala Ala Gln Leu Ala Ala Ser Gln Ala Val Tyr Val 20 25 30 Trp Thr Thr Asp Val Pro Thr Leu Pro Gly Val Pro Leu Ala Thr Asp 35 40 45 Val Pro Lys Asn Asp Glu Pro Thr Ile Ala Trp Phe Gly Ile Leu Ile 50 55 60 Gly Val Gly Leu Ala Ile Val Arg Asn Ala Leu Thr Val Lys Leu Gly 65 70 75 80 Gly Val Asp Arg Gly Glu Leu Asp Thr Pro Arg Ala Glu Tyr Glu Thr 85 90 95 Ala Leu Ala Glu Cys Asp Ala Ile Glu Leu Ser Thr Ala Lys Ile Val 100 105 110 Ala Glu His Gly Val His Ser Gly Gly Asn Ile Phe Glu Arg Ile Val 115 120 125 Gly Asp Val Glu Asn Ala Val Ala Ala Ala Glu Arg Asp Val His Leu 130 135 140 Ala Leu Leu Gln Gly Tyr Lys Glu Arg Leu Glu Asp Leu Met Lys Val 145 150 155 160 Asp Glu Ala Glu Val Ala Gly Leu Gly Ser Lys Thr Pro Arg Ser Leu 165 170 175 Asp Ala Tyr Arg Ala Leu Phe Ala Thr Leu Pro Val Pro Gly Ile Ser 180 185 190 Tyr Met Phe Gln Asp Asp Lys Glu Phe Ala Arg Leu Arg Val Gln Gly 195 200 205 Pro Asn Cys Met Leu Ile Ala Ala Val Glu Gly Ala Leu Pro Ala Asn 210 215 220 Phe Pro Leu Ser Glu Lys Ala Tyr Ala Ala Val Val Asn Gly Asp Thr 225 230 235 240 Leu Ala Ala Ala Leu Ala Asp Gly Arg Leu Phe Met Leu Asp Tyr Lys 245 250 255 Pro Leu Ala Ile Leu Asp Pro Gly Thr Tyr Gly Gly Glu Ala Lys Tyr 260 265 270 Val Ser Gln Pro Met Ala Leu Phe Ala Val Pro Pro Gly Gly Ala Ser 275 280 285 Leu Ile Pro Val Ala Ile Gln Cys Gly Gln Asp Pro Ala Asp Cys Pro 290 295 300 Ile Phe Thr Pro Ser Pro Ala Ala Asp Arg Gln Trp Gly Trp Glu Met 305 310 315 320 Ala Lys Phe Val Val Gln Val Ala Asp Gly Asn Tyr His Glu Leu Phe 325 330 335 Ala His Leu Ala Arg Thr His Leu Val Ile Glu Ala Phe Ala Val Ala 340 345 350 Thr His Arg His Leu Ala Glu Ala His Pro Val Trp Ala Leu Leu Val 355 360 365 Pro His Phe Glu Gly Thr Leu Phe Ile Asn Glu Gln Ala Ala Thr Ser 370 375 380 Leu Ile Ala Ala Asn Gly Pro Ile Asp His Ile Phe Ala Gly Thr Ile 385 390 395 400 Ala Ser Ser Gln Leu Ala Ala Val Asp Ala Arg Leu Ala Phe Asp Phe 405 410 415 Arg Gly Lys Met Pro His Ala Asp Phe Ala Ala Arg Gly Val Gly Val 420 425 430 Asp Ser Ala Leu Ala Asp Tyr Pro Tyr Arg Asp Asp Ala Leu Leu Val 435 440 445 Trp Asp Ala Ile His Glu Trp Ala Arg Gln Tyr Val Asp Leu Tyr Tyr 450 455 460 Thr Gly Asp Thr Asp Ile Val Ala Asp Thr Glu Leu Ala Ala Trp Ala 465 470 475 480 Ala Cys Leu Ala Gly Glu Ala Lys Val Gly Gly Leu Gly Pro Val Thr 485 490 495 Thr Arg Asn Gln Leu Ala Glu Ile Cys Ala Met Val Met Phe Thr Ala 500 505 510 Ser Ala Gln His Ala Ala Val Asn Leu Pro Gln Lys Asp Ile Met Ala 515 520 525 Phe Ala Pro Ala Val Thr Gly Ala Ala Trp Gln Pro Ala Pro Asn Gly 530 535 540 Gln Arg Gly His Asp Lys Thr Gly Trp Leu Ala Met Met Pro Pro Met 545 550 555 560 Ala Leu Ala Leu Glu Gln Leu Asn Val Leu Glu Leu Leu Gly Ser Val 565 570 575 His Tyr Arg Pro Leu Gly Asp Tyr Arg Ser Asn Ala Phe Pro Tyr Pro 580 585 590 Gln Trp Phe Gln Asp Pro Arg Val Thr Ala Ala Glu Gly Pro Leu Ala 595 600 605 Trp Phe Gln Ala Ala Leu Ala Asp Val Glu Ala Glu Ile Val Thr Arg 610 615 620 Asn Ala Glu Arg Met Gln Pro Tyr Pro Tyr Leu Gln Pro Ser Leu Ile 625 630 635 640 Pro Thr Ser Ile Asn Ile 645 <210> 2 <211> 646 <212> PRT <213> artificial sequence <220> <223> V569F mutant of 9-lipoxygenase <400> 2 Met Pro Ser Ala Asn Pro Ser Leu Pro Gln Asn Asp Thr Pro Ala Glu 1 5 10 15 Gln Ala Ala Arg Ala Ala Gln Leu Ala Ala Ser Gln Ala Val Tyr Val 20 25 30 Trp Thr Thr Asp Val Pro Thr Leu Pro Gly Val Pro Leu Ala Thr Asp 35 40 45 Val Pro Lys Asn Asp Glu Pro Thr Ile Ala Trp Phe Gly Ile Leu Ile 50 55 60 Gly Val Gly Leu Ala Ile Val Arg Asn Ala Leu Thr Val Lys Leu Gly 65 70 75 80 Gly Val Asp Arg Gly Glu Leu Asp Thr Pro Arg Ala Glu Tyr Glu Thr 85 90 95 Ala Leu Ala Glu Cys Asp Ala Ile Glu Leu Ser Thr Ala Lys Ile Val 100 105 110 Ala Glu His Gly Val His Ser Gly Gly Asn Ile Phe Glu Arg Ile Val 115 120 125 Gly Asp Val Glu Asn Ala Val Ala Ala Ala Glu Arg Asp Val His Leu 130 135 140 Ala Leu Leu Gln Gly Tyr Lys Glu Arg Leu Glu Asp Leu Met Lys Val 145 150 155 160 Asp Glu Ala Glu Val Ala Gly Leu Gly Ser Lys Thr Pro Arg Ser Leu 165 170 175 Asp Ala Tyr Arg Ala Leu Phe Ala Thr Leu Pro Val Pro Gly Ile Ser 180 185 190 Tyr Met Phe Gln Asp Asp Lys Glu Phe Ala Arg Leu Arg Val Gln Gly 195 200 205 Pro Asn Cys Met Leu Ile Ala Ala Val Glu Gly Ala Leu Pro Ala Asn 210 215 220 Phe Pro Leu Ser Glu Lys Ala Tyr Ala Ala Val Val Asn Gly Asp Thr 225 230 235 240 Leu Ala Ala Ala Leu Ala Asp Gly Arg Leu Phe Met Leu Asp Tyr Lys 245 250 255 Pro Leu Ala Ile Leu Asp Pro Gly Thr Tyr Gly Gly Glu Ala Lys Tyr 260 265 270 Val Ser Gln Pro Met Ala Leu Phe Ala Val Pro Pro Gly Gly Ala Ser 275 280 285 Leu Ile Pro Val Ala Ile Gln Cys Gly Gln Asp Pro Ala Asp Cys Pro 290 295 300 Ile Phe Thr Pro Ser Pro Ala Ala Asp Arg Gln Trp Gly Trp Glu Met 305 310 315 320 Ala Lys Phe Val Val Gln Val Ala Asp Gly Asn Tyr His Glu Leu Phe 325 330 335 Ala His Leu Ala Arg Thr His Leu Val Ile Glu Ala Phe Ala Val Ala 340 345 350 Thr His Arg His Leu Ala Glu Ala His Pro Val Trp Ala Leu Leu Val 355 360 365 Pro His Phe Glu Gly Thr Leu Phe Ile Asn Glu Gln Ala Ala Thr Ser 370 375 380 Leu Ile Ala Ala Asn Gly Pro Ile Asp His Ile Phe Ala Gly Thr Ile 385 390 395 400 Ala Ser Ser Gln Leu Ala Ala Val Asp Ala Arg Leu Ala Phe Asp Phe 405 410 415 Arg Gly Lys Met Pro His Ala Asp Phe Ala Ala Arg Gly Val Gly Val 420 425 430 Asp Ser Ala Leu Ala Asp Tyr Pro Tyr Arg Asp Asp Ala Leu Leu Val 435 440 445 Trp Asp Ala Ile His Glu Trp Ala Arg Gln Tyr Val Asp Leu Tyr Tyr 450 455 460 Thr Gly Asp Thr Asp Ile Val Ala Asp Thr Glu Leu Ala Ala Trp Ala 465 470 475 480 Ala Cys Leu Ala Gly Glu Ala Lys Val Gly Gly Leu Gly Pro Val Thr 485 490 495 Thr Arg Asn Gln Leu Ala Glu Ile Cys Ala Met Val Met Phe Thr Ala 500 505 510 Ser Ala Gln His Ala Ala Val Asn Leu Pro Gln Lys Asp Ile Met Ala 515 520 525 Phe Ala Pro Ala Val Thr Gly Ala Ala Trp Gln Pro Ala Pro Asn Gly 530 535 540 Gln Arg Gly His Asp Lys Thr Gly Trp Leu Ala Met Met Pro Pro Met 545 550 555 560 Ala Leu Ala Leu Glu Gln Leu Asn Phe Leu Glu Leu Leu Gly Ser Val 565 570 575 His Tyr Arg Pro Leu Gly Asp Tyr Arg Ser Asn Ala Phe Pro Tyr Pro 580 585 590 Gln Trp Phe Gln Asp Pro Arg Val Thr Ala Ala Glu Gly Pro Leu Ala 595 600 605 Trp Phe Gln Ala Ala Leu Ala Asp Val Glu Ala Glu Ile Val Thr Arg 610 615 620 Asn Ala Glu Arg Met Gln Pro Tyr Pro Tyr Leu Gln Pro Ser Leu Ile 625 630 635 640 Pro Thr Ser Ile Asn Ile 645 <210> 3 <211> 1941 <212> DNA <213> Sphingomonas macrogoltabidus <400> 3 ttgccatcgg ccaatcccag cctgccgcaa aacgatacgc ccgccgagca agcggcgcgc 60 gcggcacagc tcgccgcttc gcaggccgtc tatgtctgga ccaccgacgt cccgacgctg 120 cccggcgtgc cgctcgccac cgacgtgccg aagaacgacg aaccgacgat cgcctggttc 180 ggcatcttga tcggcgtcgg cctcgcgatc gtgcgcaatg cgctgacggt gaagctcggc 240 ggcgtcgacc ggggcgagct cgacacgccg cgcgccgaat atgaaaccgc gctcgccgag 300 360 ggcaatatct tcgaacgcat cgtcggcgac gtggaaaatg ccgtcgccgc ggctgagcgc 420 gacgtgcatc tcgctttgct tcagggatat aaggagcggc tcgaggatct catgaaggtc 480 gacgaggccg aggtggcggg gctgggcagc aagacgccgc gcagcctcga cgcatatcgc 540 gcgctgttcg ctaccctgcc ggtgccgggc atcagctata tgttccagga cgacaaggag 600 ttcgcccgcc tgcgcgtgca ggggccgaac tgcatgctga tcgcggcggt cgagggagcg 660 ctgcccgcca atttcccgct gagcgaaaag gcttacgccg cggtggtgaa tggcgacacg 720 ctggccgccg cgctcgccga cggccgcctc ttcatgctcg attacaagcc gctcgcgatc 780 ctcgaccccg gcacctatgg cggcgaagcc aaatatgtct cgcagccgat ggcgctgttc 840 gcggtaccgc cgggcggcgc atcgctcatc cccgtcgcga tccagtgcgg ccaggacccc 900 gccgactgtc cgattttcac gccctcaccg gcggccgaca ggcaatgggg ctgggaaatg 960 gcgaaattcg tcgtgcaggt cgccgacggt aattatcatg agctcttcgc ccatctggca 1020 cgcacccatc tggtgatcga ggcgttcgcg gtcgcgacgc accgccatct tgccgaggcg 1080 catccggtgt gggcgctgct cgtcccgcat ttcgagggga cattgttcat caacgaacag 1140 gcggcgacct cgctgatcgc ggcgaacggc ccgatcgacc atattttcgc ggggacgatc 1200 gcgtcgagcc agcttgccgc agtcgatgcg cggctggcgt tcgatttccg gggcaagatg 1260 ccgcacgccg attttgccgc gcggggcgtc ggggtcgatt cggcgctcgc cgactatccg 1320 tatcgcgacg acgcgctgct ggtgtgggac gcgatccacg aatgggcgcg gcaatatgtc 1380 gatctttatt atacgggcga caccgatatc gtcgccgata ccgagctggc ggcatgggcc 1440 gcgtgcctcg cgggcgaagc caaggtcggc gggctcggcc cggtgacgac gcgcaaccag 1500 ctcgccgaaa tctgcgcgat ggtgatgttc accgccagcg cgcagcatgc ggcggtcaat 1560 ttaccgcaaa aggacatcat ggccttcgcc cccgcggtga ccggcgcagc gtggcaaccg 1620 gcgccgaacg gccagcgcgg acacgacaag acgggctggc tcgcgatgat gccgccgatg 1680 gccctcgcgc tcgaacaatt gaacgtgctc gaactgctgg ggtcggtgca ttatcgcccg 1740 ctcggcgact atcgcagcaa cgcctttccc tatccgcagt ggttccagga tccgcgcgtc 1800 accgcggcgg aggggcccgct cgcctggttt caggcggcgc ttgccgacgt cgaggcggaa 1860 atcgtcacgc gcaatgccga gcggatgcag ccctatccct atctgcagcc gagcctgatc 1920 ccgacgagca tcaatatctg a 1941 <210> 4 <211> 1941 <212> DNA <213> artificial sequence <220> <223> V569F mutant of 9-lipoxygenase <400> 4 ttgccatcgg ccaatcccag cctgccgcaa aacgatacgc ccgccgagca agcggcgcgc 60 gcggcacagc tcgccgcttc gcaggccgtc tatgtctgga ccaccgacgt cccgacgctg 120 cccggcgtgc cgctcgccac cgacgtgccg aagaacgacg aaccgacgat cgcctggttc 180 ggcatcttga tcggcgtcgg cctcgcgatc gtgcgcaatg cgctgacggt gaagctcggc 240 ggcgtcgacc ggggcgagct cgacacgccg cgcgccgaat atgaaaccgc gctcgccgag 300 360 ggcaatatct tcgaacgcat cgtcggcgac gtggaaaatg ccgtcgccgc ggctgagcgc 420 gacgtgcatc tcgctttgct tcagggatat aaggagcggc tcgaggatct catgaaggtc 480 gacgaggccg aggtggcggg gctgggcagc aagacgccgc gcagcctcga cgcatatcgc 540 gcgctgttcg ctaccctgcc ggtgccgggc atcagctata tgttccagga cgacaaggag 600 ttcgcccgcc tgcgcgtgca ggggccgaac tgcatgctga tcgcggcggt cgagggagcg 660 ctgcccgcca atttcccgct gagcgaaaag gcttacgccg cggtggtgaa tggcgacacg 720 ctggccgccg cgctcgccga cggccgcctc ttcatgctcg attacaagcc gctcgcgatc 780 ctcgaccccg gcacctatgg cggcgaagcc aaatatgtct cgcagccgat ggcgctgttc 840 gcggtaccgc cgggcggcgc atcgctcatc cccgtcgcga tccagtgcgg ccaggacccc 900 gccgactgtc cgattttcac gccctcaccg gcggccgaca ggcaatgggg ctgggaaatg 960 gcgaaattcg tcgtgcaggt cgccgacggt aattatcatg agctcttcgc ccatctggca 1020 cgcacccatc tggtgatcga ggcgttcgcg gtcgcgacgc accgccatct tgccgaggcg 1080 catccggtgt gggcgctgct cgtcccgcat ttcgagggga cattgttcat caacgaacag 1140 gcggcgacct cgctgatcgc ggcgaacggc ccgatcgacc atattttcgc ggggacgatc 1200 gcgtcgagcc agcttgccgc agtcgatgcg cggctggcgt tcgatttccg gggcaagatg 1260 ccgcacgccg attttgccgc gcggggcgtc ggggtcgatt cggcgctcgc cgactatccg 1320 tatcgcgacg acgcgctgct ggtgtgggac gcgatccacg aatgggcgcg gcaatatgtc 1380 gatctttatt atacgggcga caccgatatc gtcgccgata ccgagctggc ggcatgggcc 1440 gcgtgcctcg cgggcgaagc caaggtcggc gggctcggcc cggtgacgac gcgcaaccag 1500 ctcgccgaaa tctgcgcgat ggtgatgttc accgccagcg cgcagcatgc ggcggtcaat 1560 ttaccgcaaa aggacatcat ggccttcgcc cccgcggtga ccggcgcagc gtggcaaccg 1620 gcgccgaacg gccagcgcgg acacgacaag acgggctggc tcgcgatgat gccgccgatg 1680 gccctcgcgc tcgaacaatt gaacttcctc gaactgctgg ggtcggtgca ttatcgcccg 1740 ctcggcgact atcgcagcaa cgcctttccc tatccgcagt ggttccagga tccgcgcgtc 1800 accgcggcgg aggggcccgct cgcctggttt caggcggcgc ttgccgacgt cgaggcggaa 1860 atcgtcacgc gcaatgccga gcggatgcag ccctatccct atctgcagcc gagcctgatc 1920 ccgacgagca tcaatatctg a 1941

Claims (10)

삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 하기 화학식 8로 표시되는, 신규 이수산화지방산:
[화학식 8]
Figure 112023028428227-pat00023
.
A novel dihydroxide fatty acid represented by Formula 8:
[Formula 8]
Figure 112023028428227-pat00023
.
 제6항에 있어서,
상기 [화학식 8]의 신규 이수산화지방산은 스핑고모나스 마크로골타비두스(Sphingomonas macrogoltabidus) 유래 9-리폭시게나아제; 또는 상기 9-리폭시게나아제의 아미노산 서열로부터, 569번째 아미노산인 발린(V)을 페닐알라닌(F)로 치환시킨 9-리폭시게나아제 변이체를 유효성분으로 포함하는 이수산화지방산 제조용 조성물을
도코사헥사엔산(docosahexaenoic acid)을 기질로 하여 처리하는 단계를 포함하는 이수산화지방산 제조방법으로 제조된 것을 특징으로 하는 신규 이수산화지방산.
According to claim 6,
The novel dihydrogenated fatty acid of [Formula 8] is 9-lipoxygenase derived from Sphingomonas macrogoltabidus; Alternatively, from the amino acid sequence of the 9-lipoxygenase, a composition for preparing a dihydroxylated fatty acid comprising a 9-lipoxygenase variant in which valine (V), the 569th amino acid, is substituted with phenylalanine (F) as an active ingredient
A novel dihydrogenated fatty acid, characterized in that it is produced by a method for producing dihydrogenated fatty acid comprising the step of treating docosahexaenoic acid as a substrate.
 제7항에 있어서,
상기 9-리폭시게나아제는 서열번호 1의 아미노산 서열로 이루어지고, 상기 9-리폭시게나아제 변이체는 서열번호 2의 아미노산 서열로 이루어진 것인, 신규 이수산화지방산.
According to claim 7,
The 9-lipoxygenase consists of the amino acid sequence of SEQ ID NO: 1, and the 9-lipoxygenase variant consists of the amino acid sequence of SEQ ID NO: 2, a novel dihydroxylated fatty acid.
 제8항에 있어서,
상기 9-리폭시게나아제는 서열번호 3의 염기 서열로 이루어지고, 상기 9-리폭시게나아제 변이체는 서열번호 4의 염기 서열로 이루어진 것인, 신규 이수산화지방산.
According to claim 8,
The 9-lipoxygenase consists of the nucleotide sequence of SEQ ID NO: 3, and the 9-lipoxygenase variant consists of the nucleotide sequence of SEQ ID NO: 4, a novel dihydroxylated fatty acid.
 제7항에 있어서,
상기 처리는 pH 6.5~9.0 및 온도 20~40℃에서 수행되는 것인, 신규 이수산화지방산.
According to claim 7,
The treatment is carried out at a pH of 6.5 to 9.0 and a temperature of 20 to 40 ° C., a novel dihydrogenated fatty acid.
KR1020220062926A 2020-05-29 2022-05-23 Composition and method for producing hydroxy fatty acid or dihydroxy fatty acid using 9-lipoxygenase or variant thereof KR102555535B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020220062926A KR102555535B1 (en) 2020-05-29 2022-05-23 Composition and method for producing hydroxy fatty acid or dihydroxy fatty acid using 9-lipoxygenase or variant thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020200064827A KR102402601B1 (en) 2020-05-29 2020-05-29 Composition and method for producing hydroxy fatty acid or dihydroxy fatty acid using 9-lipoxygenase or variant thereof
KR1020220062926A KR102555535B1 (en) 2020-05-29 2022-05-23 Composition and method for producing hydroxy fatty acid or dihydroxy fatty acid using 9-lipoxygenase or variant thereof

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
KR1020200064827A Division KR102402601B1 (en) 2020-05-29 2020-05-29 Composition and method for producing hydroxy fatty acid or dihydroxy fatty acid using 9-lipoxygenase or variant thereof

Publications (2)

Publication Number Publication Date
KR20220075286A KR20220075286A (en) 2022-06-08
KR102555535B1 true KR102555535B1 (en) 2023-07-12

Family

ID=78868367

Family Applications (2)

Application Number Title Priority Date Filing Date
KR1020200064827A KR102402601B1 (en) 2020-05-29 2020-05-29 Composition and method for producing hydroxy fatty acid or dihydroxy fatty acid using 9-lipoxygenase or variant thereof
KR1020220062926A KR102555535B1 (en) 2020-05-29 2022-05-23 Composition and method for producing hydroxy fatty acid or dihydroxy fatty acid using 9-lipoxygenase or variant thereof

Family Applications Before (1)

Application Number Title Priority Date Filing Date
KR1020200064827A KR102402601B1 (en) 2020-05-29 2020-05-29 Composition and method for producing hydroxy fatty acid or dihydroxy fatty acid using 9-lipoxygenase or variant thereof

Country Status (1)

Country Link
KR (2) KR102402601B1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102086235B1 (en) 2018-08-03 2020-03-06 건국대학교 산학협력단 Method for producing resolvin D2 analogues by combination reaction using recombinant lipoxigenases and resolvin D2 analogues therefrom

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102086235B1 (en) 2018-08-03 2020-03-06 건국대학교 산학협력단 Method for producing resolvin D2 analogues by combination reaction using recombinant lipoxigenases and resolvin D2 analogues therefrom

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PubChem SID: 250132961 (2015.04.28) 1부.*

Also Published As

Publication number Publication date
KR20220075286A (en) 2022-06-08
KR20210147497A (en) 2021-12-07
KR102402601B1 (en) 2022-05-25

Similar Documents

Publication Publication Date Title
KR100224120B1 (en) Gene for fatty acid desaturase, vector containing said gene
KR101934900B1 (en) Composition for producing hydroxyfatty acid, hepoxilin b or trioxilin b
KR101642583B1 (en) Method for the production of 13-hydroxylinoleic acid by lipoxygenase from Burkholderia thailandensis and composition therefor
KR101934904B1 (en) Composition for producing 11-hydroxyfatty acid, hepoxilin d3 or trioxilin d3
KR102086235B1 (en) Method for producing resolvin D2 analogues by combination reaction using recombinant lipoxigenases and resolvin D2 analogues therefrom
KR102555535B1 (en) Composition and method for producing hydroxy fatty acid or dihydroxy fatty acid using 9-lipoxygenase or variant thereof
JP2003052357A (en) Marine microorganism, and method for manufacturing carotenoid pigment and/or higher unsaturated fatty acid by using the microorganism
KR20090088630A (en) Amino acid deaminase gene of proteus mirabilis, protein, recombinant vector and transformant, and method for manufacturing functional organic acids using the same
JP4221476B2 (en) Plasmid cloned icosapentaenoic acid biosynthesis genes and cyanobacteria producing icosapentaenoic acid
KR102379220B1 (en) Composition and method for producing hydroxy fatty acid or dihydroxy fatty acid using 5r-lipoxygenase variant
KR102120000B1 (en) Production of 5,15-dihydroxy arachidonic acid by new bacterial lipoxygenase
KR102086298B1 (en) Method for producing lipoxin analogues by combination reation using recombinant lipoxigenases and lipoxin analogues therefrom
KR102086236B1 (en) Method for producing 8,15-dihydroperoxy fatty acid or leukotriene analogues by combination reation using recombinant lipoxigenases
KR102184967B1 (en) Composition for producing hydroxyfatty acid, hepoxilin b analog or trioxilin b analog
JP2000228987A (en) Production of coenzyme q10
KR102278582B1 (en) Composition for producing dihydroxy fatty acid comprising lipoxygenase variant and method for producing dihydroxy fatty acid using the same
KR102286087B1 (en) Chlamydomonas mutants and use thereof
KR102131549B1 (en) Production of 5,15-dihydroxy eicosapentaenoic acid by new bacterial lipoxygenase
KR102442480B1 (en) Composition for culturing chlamydomonas mutants and culture method thereof
KR102131550B1 (en) Production of resolvin d5 by new bacterial lipoxygenase
KR102190311B1 (en) Method for the production of the protectin DX by combination reaction of arachidonate mouse 8-lipoxygenase and microbial arachidonate 15-lipoxygenase
KR20240029670A (en) Novel 15R-lipoxygenase, composition and method for producing hydroxylated fatty acid or dihydroxy fatty acid using the same
TW200407430A (en) Cells secreting R-α- lipoic acid and method for fermentative production of said R-α- lipoic acid
KR20240094252A (en) Method for producing novel hydroxylated fatty acids
JP4276886B2 (en) Enzyme having interconversion action between scyllo-inosose and D-kilo-1-inosose and its production and application

Legal Events

Date Code Title Description
A107 Divisional application of patent
E902 Notification of reason for refusal
E701 Decision to grant or registration of patent right
GRNT Written decision to grant