KR101642583B1 - Method for the production of 13-hydroxylinoleic acid by lipoxygenase from Burkholderia thailandensis and composition therefor - Google Patents

Abstract

The present invention relates to a process for producing 13-hydroxyxylic acid and a composition thereof, and more particularly, Member kolde Syrian den tailran sheath (Burkholderia thailandensis) to react at the optimized linoleate acid (linoleic acid) reaction conditions by using a hydroxide synthase of bacterial origin (Lipoxygenase) and to optimize the conditions and the 13-hydroxide linoleate acid yield It is the method of production.
According to the present invention, 13-hydroxide ricinoleic acid can be produced with high yield by a high specificity and environmentally friendly method, and thus produced 13-hydroxide ricinoleic acid can be used as a raw material in various industrial fields.

Description

TECHNICAL FIELD The present invention relates to a method for preparing 13-hydroxylolynoic acid from lipoxygenase from Burkholderia thailandensis and composition therefor,

The present invention relates to a process for preparing 13-hydroxylinoleic acid using lipoxygenase and a composition thereof, and more particularly to a process for preparing 13-hydroxylinoleic acid from linoleic acid by using 13-hydroxy linoleic acid Hydroxynonoleic acid in a high yield by using an enzyme converting the enzyme and a composition thereof.

Hydroxylated fatty acids are substances that exist in nature and exist in plants, insects and lipids existing in microorganisms, and they are also found in waxes, triglycerol, ceresbrocide and the like. There are three types of hydroxylated fatty acids that enzymes present in microorganisms can produce through bioconversion of unsaturated fatty acids: hydroxylation, dihydroxylation, and trihydroxy fatty acids. Hydroxylated fatty acid has a hydroxyl group, so it reacts well with other fatty acids and is used as an industrial starting material for waxes, nylons, resins, lubricants, coatings and plastics. It has antifungal activity and reduces surface tension. It can also be used as raw material. It also has important industrial value as a precursor of the typical lactone of natural fragrance.

Lipoxygenase, which synthesizes hydroxylated fatty acids, is a diazotizing enzyme that requires iron to proceed with the reaction. And catalyzes stereospecificity and reaction specificity in the diazotization reaction of polyunsaturated fatty acids having one or more cis, cis-1,4 pentadiene. In particular, 13-lipoxygenase, which attaches a hydroxyl group at position 13, is a non-heme iron enzyme consisting of 896-941 amino acids and specifically forms hydroxyl at position 13 only. Examples of microorganisms that produce 13-hydroxynonoleic acid in the past include Gaeumannomyces graminis tritici , Nostoc punctiforme) and Pseudomonas Erotic not labor (Pseudomonas aeruginosa .

To date, 13-hydroxynonoleic acid has been produced from linoleic acid using soybean 13-lipoxygenase. However, since the yield of enzyme purification was very low and the enzyme activity was not high, it was difficult to mass-produce 13-hydroxy linoleic acid.

[Prior Patent Literature]

Japanese Laid-Open Patent Publication No. 2002-315593

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above problems, and it is an object of the present invention to provide a process for producing 13-hydroxynonoleic acid in a high yield without producing by-products in an environmentally friendly process.

Another object of the present invention is to provide a composition which can produce 13-hydroxynolinoleic acid in a high yield without producing any by-products in an environmentally friendly process.

In order to accomplish the above object, the present invention relates to a method for producing Burkholderia The present invention provides a composition for the production of 13-hydroxylinoleic acid, which comprises as an active ingredient a lipoic oxidase derived from L. thailandensis .

In one embodiment of the present invention, the lipid oxidizing enzyme derived from Burkoleria tylandercis is preferably composed of the amino acid sequence of SEQ ID NO: 1, but the present invention is not limited thereto. All of the mutant enzymes capable of achieving the effect of the present invention are included in the scope of the present invention.

In another embodiment of the present invention, the composition preferably further comprises linoleic acid as a substrate, but is not limited thereto.

The present invention also provides a composition for the production of 13-hydroxylinoleic acid comprising a gene coding for a lipid oxidation enzyme derived from Burkoleria tylandense as an active ingredient.

In one embodiment of the present invention, the gene coding for the lipolytic enzyme derived from Bacillardia tylendansis is preferably composed of the nucleotide sequence of SEQ ID NO: 2, All mutant genes capable of achieving the desired effect of the invention are included within the scope of the present invention.

In addition, the present invention provides a method for producing 13-hydroxylinoleic acid by biotransformation by reacting an unsaturated fatty acid-producing enzyme with an enzyme producing a hydroxylated fatty acid.

In an embodiment of the present invention, the enzyme producing the hydroxycarboxylic acid from the unsaturated fatty acid is preferably derived from a bacteriolytic agent, and the enzyme producing the hydroxycarboxylic acid from the unsaturated fatty acid is preferably a lipid oxidizing enzyme But not limited to this.

In another embodiment of the present invention, the substrate is preferably linoleic acid and the method comprises using linoleic acid in a range from 5 g / l to 25 g / l as substrate But is not limited thereto.

In another embodiment of the present invention, the method is preferably but not limited to adding methanol as an organic solvent.

In one embodiment of the present invention, the process is preferably carried out at a pH in the range of from 7.0 to 8.0, and the method is preferably, but not limited to, performing the reaction at a temperature in the range of from 20 캜 to 35 캜.

In another embodiment of the present invention, the method preferably treats the concentration of the lipid oxidizing enzyme, which is an enzyme that produces the hydroxylated fatty acid from the unsaturated fatty acid, in the range of 1.5 g / l to 3.0 g / l, but is not limited thereto.

Hereinafter, the present invention will be described.

In order to develop a more effective method for producing 13-hydroxyoctadecadienoic acid (13-hydroxy-9,11 ( Z , E ) -octadecadienoic acid) using lipid oxidation enzyme, Burkholderia The present invention has been accomplished based on this finding that 13-hydroxynonoleic acid can be produced in high yield without producing any by-products by this environmentally friendly process.

The present inventors have investigated an enzyme capable of converting an unsaturated fatty acid, linoleic acid, into a hydroxylated fatty acid, and selected an enzyme capable of producing 13-hydroxylated linoleic acid in high yield.

Burkholderia thailandensis ) was constructed, and a large amount of lipid oxidation enzymes were prepared using these enzymes, and the optimal reaction conditions for obtaining 13-hydroxy linoleic acid in an environmentally friendly process with high yield were confirmed.

The present invention provides the use of lipoxygenase for the production of 13-hydroxylinoleic acid.

The present invention also provides a recombinant microorganism having a gene of lipoxygenase used for production of 13-hydroxylinoleic acid.

Specifically, it is possible to prepare 13-hydroxy linoleic acid by using linoleic acid as a substrate And provides a lipid oxidation enzyme derived from Burkholderia thailandensis strain.

The present invention also provides a recombinant expression vector comprising a lipid oxidation enzyme.

Specifically, the present invention provides Provides a recombinant expression vector pET 28a (+) / lipooxidase derived from Burkholderia thailandensis strain and containing a lipid oxidase gene.

The present invention also provides a microorganism transformed with a recombinant expression vector comprising a lipid oxidizing enzyme. Burkholderia thailandensis ) (ER) 2566 pET 28a (+) / lipooxidase transformed with the recombinant expression vector containing the lipid oxidase gene derived from the strain.

In the present invention, any recombinant expression vector can be used as long as it can be used for the production of 13-hydroxynonoleic acid. Any vector can be used for the recombinant method including the expression vector pET-28a (+), Escherichia coli ER 2566 is preferably used as the microorganism to be transformed, but any strain that can produce an enzyme protein overexpressing the desired gene and transforming with a recombinant expression vector and producing an active enzyme protein can be used.

The present invention also provides a recombinant expression vector comprising (1) a recombinant expression vector comprising a lipid oxidase gene; (2) culturing the microorganism transformed with the recombinant expression vector; (3) inducing expression of the hydroxylated synthase gene, and (4) providing an expressed recombinant protein and an enzyme containing the expressed recombinant protein.

The present invention provides a production method for obtaining 13-hydroxynonoleic acid in high yield using cultured lipid oxidation enzymes.

Specifically, the present invention provides a method for producing 13-hydroxynonoleic acid using the above-mentioned lipid oxidase capable of selectively synthesizing only 13-hydroxide ricinoleic acid without producing any by-products.

It is preferable that the lipoic oxidase is cultured in the E. coli transformed with the recombinant expression vector containing the lipid oxidase gene to induce the expression of the recombinant enzyme gene and recover the expressed protein.

In addition, it is preferable to use a fatty acid as a substrate, and more preferably to use linoleic acid. The concentration of the substrate is preferably in the range of 5 g / l to 25 g / l, more preferably 20 g / l or less.

In addition, the enzyme reaction is preferably performed in a pH range of 7.0 to 8.0, more preferably in a pH range of about 7.5.

The enzyme reaction is preferably performed at a temperature in the range of 20 ° C to 35 ° C, and more preferably in the temperature range of about 25 ° C or less.

In addition, the time of the enzyme reaction can be appropriately controlled by a conventional method.

As can be seen from the present invention, the present invention provides a production method for obtaining 13-hydroxynonoleic acid in high yield using a recombinant expression vector containing a lipid oxidase gene and an expressed protein.

The Burkholderia < RTI ID = 0.0 > thailandensis ) Can produce 13-hydroxynonoleic acid, a raw material for industrial products, with high yield and other byproducts than the papers reported so far, with high specificity and environmentally friendly method.

FIG. 1A shows the effect of the lipid oxidation enzyme of the present invention on 13-hydroxynonoleic acid production activity (●: HEPES buffer, ○: Tris-HCl buffer)
Fig. 1 (b) shows the effect of the temperature of lipid oxidation enzyme on 13-hydroxynonoleic acid production activity, and Fig. 1 (c) shows the thermal stability of 13-hydroxynonoleic acid production activity of hydroxycarboxylase.
FIG. 2A shows the results of the type of metal ion in the production of 13-hydroxynonoleic acid of the lipid oxidizing enzyme of the present invention, and FIG. 2B shows the result of the concentration of the metal ion in the production of linoleic acid.
FIG. 3A shows the results of the type of the organic solvent in the production of the 13-hydroxynaphthoic acid of the lipid oxidizing enzyme of the present invention, and FIG. 3B shows the results in accordance with the concentration of the organic solvent.
Fig. 4A shows the optimum enzyme concentration (?: Linoleic acid,?: 13-linolenic acid) in producing 13-hydroxynonoleic acid of the lipid oxidizing enzyme of the present invention, Fig. 4B shows the concentration : Conversion rate, and?: 13-linolenic acid hydroxide).
Figure 5 is a graphical representation of the inventive Burkholderia < RTI ID = 0.0 > thailandensis ) shows the hourly production of 13-hydroxynaphthoic acid using the lipid oxidation enzyme derived from KCTC 23190. (●: 13-Lynol oxidase from Bacolodiola tylandensech) ■: Lynolate acid (Lactic acid from Bacolodiola tylandensis) ○: 13-Lynol oxidase from soybean Oxidase) □: Linoleic acid (soybean-derived fatty oxidase)
Figure 6 is a graphical representation of the results of the Burkholderia < RTI ID = 0.0 > thailandensis ) KCTC 23190, which is a recombinant vector.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are for illustrative purposes only, and it will be apparent to those skilled in the art that the scope of the present invention is not construed as being limited by these examples.

Example  One. Fatty oxidase  Production of Recombinant Expression Vectors and Proteins Containing Gene

To prepare the lipid oxidase gene of the present invention, Burkholderia thailandensis ) was first isolated.

Specifically, when the nucleotide sequence and the amino acid sequence are already specified Burkholderia In order to carry out polymerase chain reaction (PCR) on the basis of the DNA sequence of the lipid oxidation enzyme derived from thailandensis KCTC 23190 (microorganism resource center, Daejeon), strains were selected, and Bacillardia tylalan Genomic DNA of Burkholderia thailandensis was extracted and used as a template for PCR. A primer based on the DNA sequence of lipid oxidase was designed (Forward: 5'- GCTAGC ATGGTCAATCACAAAACCGG- 3 '(SEQ ID NO: 3) / Reverse: 5'- AAGCTT TCAAATGTTCGTGCTTGCCG-3' (SEQ ID NO: 4)). Nhe I and Hind III were used as restriction enzymes. Recombinant plasmid vector pET 28a (+) (manufactured by Novagen) was also prepared by restriction enzyme treatment and ligation to produce pET 28 (+) a / lipid oxidase.

The recombinant expression vector thus obtained was transformed into Escherichia coli ER 2566 strain purchased from New England Biolabs (Hertfordshire, UK) by a conventional transformation method, and the transformed microorganism was added with 20% glycerine solution Lt; RTI ID = 0.0 > -70 C < / RTI > prior to culturing for the production of 13-hydroxynonoleic acid.

Example  2. Fatty oxidase  Manufacturing and recovery

Recombinant E. coli for protein expression of the enzyme was cultured in a 500 ml LB (Difco, Sparks, MD, USA) medium and a flask with 20 μg / ml kanamycin under aeration conditions of 250 rev / min at 37 ° C Lt; / RTI > When the absorbance of the bacteria reached from 0.6 to 0.8 at 600 nm, a final concentration of 0.1 mM IPTG was added to induce lipid oxidase expression, and the culture was incubated for 16 hours at 16 ° C with stirring at 150 rev / min .

Escherichia coli cells containing cultured lipid oxidation enzymes were collected and used. The overexpressed and overexpressed lipid oxidase was centrifuged at 6,000 x g for 30 minutes at 4 ° C, washed twice with 0.85% sodium chloride (NaCl), and then lysed with 13- Were used as recombinant cells for the production of Iksan.

The culture medium of the E. coli cells containing the lipid oxidation enzyme was disrupted with a sonicator, and centrifuged at 13000 x g for 10 minutes at 4 ° C. The supernatant was filtered with a 0.45 μm filter. This filtrate was used as crude extract. The purification was carried out using a His-Trap HP chromatography column (Amersham bioscience), washing the column with a 50 mM sodium phosphate (Na ₂ HPO ₄ ) solvent (pH 8.0) extensively, Imidazole was extracted at a flow rate of 1 ml / min from 10 to 250 mM. The lipid oxidation enzyme was eluted with 50 mM HEPES buffer (pH 7.5) buffer, and the obtained solution was used as a purification enzyme. All purification procedures were performed at 4 ° C.

Example  3. Fatty oxidase pH 13-Hydroxy Linoleic acid  Effect on production activity

To investigate the pH effect of the lipoic oxidase on the production of 13-hydroxynonoleic acid, 50 mM HEPES buffer (pH 6.5-7.5) was added to 0.028 g / L linoleic acid as a substrate, (Tris-HCl buffer, pH 7.0-8.5) buffer solution for 2 minutes. As a result, as shown in Fig. 1A, it was found that the optimum pH was 7.5.

Example  4. Fatty oxidase  When the temperature is 13-hydroxide Linoleic acid  Effect on production activity

To investigate the temperature effect on the production of 13-hydroxynonoleic acid of the lipoic oxidase, 0.027 g / L of linoleic acid was added at a temperature of 10-40 ° C in a 50 mM HEPES buffer pH 7.5 The reaction was carried out for 2 minutes each.

As a result, as shown in FIG. 1B, it was found that the optimum temperature was 25 ° C.

Example  5. From fatty oxidase  13-Hydroxy Linoleic acid  On production activity Thermal stability

To investigate the thermal stability of the lipoic oxidase to 13-hydroxynonoleic acid production, 0.028 g / L linoleic acid was dissolved in 50 mM HEPES buffer pH 7.5 to 20-40 ° C for 1 hour The reaction was carried out at 35 ° C for 2 minutes.

As a result, it was confirmed that the activity was maintained at 80% or more as shown in Fig. 1C.

Example  6. Fatty oxidase  Investigation of Enzyme Conversion Activity by Metal Ion Concentration and Concentration

In order to investigate the activity of producing 13-hydroxynonoleic acid from linoleic acid by using a lipid oxidizing enzyme derived from Bacilleria tylandensece, an enzyme was prepared in the same manner as in Example 2, Respectively.

In order to investigate the effects of metal ion species, the reactions were treated with 0.1 mM and 1 mM of iron, calcium, zinc, cobalt, magnesium, manganese nickel, and copper, respectively, to obtain 0.028 g / ℓ linoleic acid and 0.05 ug / The reaction was terminated by the same amount of ethyl acetate as the amount of the reaction solution, and the yield of 13-hydroxy-linoleic acid was measured to determine the relative amount of the relative amount The results are shown in FIG. 2a, and copper was found to be the most optimal.

To investigate the effect of copper concentration, a 50 mM HEPES buffer solution containing 0.028 g / L linoleic acid and 0.05 μg / mℓ of enzyme from 0 mM to 0.25 mM was reacted for 2 minutes at pH 7.5 The yield of 13-hydroxynaphthaleneic acid was measured after completion of the reaction using ethyl acetate. Relative results were shown in FIG. 2b and it was confirmed that 0.1 mM copper was the most suitable.

Example  7. Fatty oxidase  Investigation of Enzyme Conversion Activity by Organic Solvent Type and Concentration

In order to investigate the activity of producing 13-hydroxynonoleic acid from linoleic acid by using a lipid oxidizing enzyme derived from Bacilleria tylandensece, an enzyme was prepared in the same manner as in Example 2, Respectively.

To investigate the effect of organic solvent species, the reactions were treated with 2% and 6% methanol, ethanol, pentane, ethyl acetate, isopropanol, dimethylsulfoxide, isooctane, butanol, The reaction was terminated by the same amount of ethyl acetate as the amount of the reaction solution, and then the reaction was terminated. The reaction was terminated by the addition of 50 mM of hepta-phosphate buffer solution containing 0.05 mol / The yield of linoleic acid hydroxide was measured and its relative results are shown in Figure 3a and methanol was found to be the most optimal.

To investigate the effect of methanol concentration, a 50 mM HEPES buffer containing 0.028 g / L linoleic acid and 0.05 μg / mℓ of enzyme in the range of 0% to 10% was reacted for 2 minutes at pH 7.5 The yield of 13-hydroxynaphthaleneic acid was measured after completion of the reaction using ethyl acetate. Relative results are shown in FIG. 3b, and it was confirmed that the optimum result was obtained when 6% of methanol was treated.

Example  8. Fatty oxidase  Concentration and Linoleic acid  Investigation of bioconversion activity on concentration change

In order to investigate the activity of producing 13-hydroxynonoleic acid from linoleic acid by using a lipid oxidizing enzyme derived from Bacilleria tylandensece, an enzyme was prepared in the same manner as in Example 2, Respectively.

In the present invention, in order to investigate the conditions showing the highest production rate of 13-hydroxynolinoleic acid when compared with the conventional conversion from linoleic acid by using lipid oxidation enzyme derived from Burkoleria tylandercis, various enzyme concentrations And substrates were reacted with the enzyme and substrate and the yield of 13-hydroxynonoleic acid was compared.

In order to investigate the effect of lipid peroxidase from Burkoleria tylandercis on the concentration of lipid peroxidase, 0 g of L-linoleic acid, 0.1 mM copper, 6% methanol and pH 7.5, / l to 3 g / l. At this time, the reaction temperature was 25 ° C and the reaction time was adjusted to 60 minutes. The reaction was then terminated by the addition of ethyl acetate and the amount of 13-hydroxynonoleic acid produced was measured. The results are shown in FIG. 4A and showed maximum conversion at an enzyme concentration of 2.5 g / l.

In order to investigate the effect of linoleic acid concentration, the reaction was carried out in the presence of 2.5 g / L enzyme, 0.1 mM copper, and 50 mM of 6% methanol in a concentration range from 5 g / l to 30 g / l of linoleic acid HEPES buffer solution was reacted at pH 7.5, ethyl acetate was added to terminate the reaction, and the yield of 13-hydroxynonoleic acid was measured. The results are shown in FIG. 4B. The maximum conversion was observed at the concentration of norepine acid.

Example  9. Fat oxidase  13-Hydroxylated Linoleic acid  production

In order to compare the production of 13-hydroxynonoleic acid with the lipid oxidase derived from the bacillary tylandercis of the present invention and the production of 13-hydroxynonoleic acid using the previously reported soybean-derived 13-lipoic oxidase , A lipid oxidizing enzyme derived from Burkholderia tylandensech was prepared by dissolving 50 mM HEPES buffer containing 2.5 g / l enzyme, 0.1 mM copper and 6% methanol at pH 7.5, 20 g / l of linoleic acid Hydroxynonoleic acid was measured as a substrate. The soybean-derived 13-lipolytic enzyme was prepared by dissolving 50 mM EPPS buffer solution containing 2.5 g / l enzyme and 0.01 mM Tween 40 at pH 8.5 , And the hourly production amount of 13-hydroxylated linoleic acid was measured using 20 g / L of linoleic acid as a substrate at a temperature of 20 ° C.

The results are shown in FIG. 5, and the lipid oxidation enzyme derived from Burkholderia tylandensech produced 20.8 g / l of 13-hydroxide linoleic acid, yielding a productivity of 10.4 g / l per hour and a conversion yield of 98.5% , 13-lipoic oxidase derived from soybean produced 13.8 g / l of 13-hydroxide linoleic acid, yielding 6.9 g / ℓ per hour and conversion yield of 65.3%. It was found that the lipid oxidation enzyme derived from Burkoleria tylandercis had 151% higher yield and 158% higher productivity than the soybean - derived lipid oxidation enzyme.

As described above, the present invention relates to a method for mass-producing a hydroxylated fatty acid which is used as a starting material in various industrial fields such as nylon, resin, wax, natural surfactant and the like using fatty oxidizing enzyme derived from Bacillardia tylendansis . Specifically, the substrate can be produced with a high yield of 13-hydroxynonoleic acid by using linoleic acid and controlling the enzyme concentration, temperature and pH by using a lipid oxidizing enzyme derived from Burkoleria tylendansis, It is possible to maximize the production efficiency while effectively reducing the production cost in the bioconversion process by using the unsaturated fatty acid contained in the plant and the oil in the raw material as well as overcoming the environment friendly condition as compared with the conventional chemical method, The production of 13-hydroxynonoleic acid is much greater than the reported soybean-derived lipid oxidase, and only 13-hydroxynonoleic acid can be obtained with high yield and high productivity.

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific descriptions are only for the preferred embodiments and that the scope of the present invention is not limited thereby. It will be obvious. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Konkuk University Industrial Cooperation Corp <120> Method for the production of 13-hydroxylinoleic acid          lipoxygenase from Burkholderia thailandensis and composition          therefor <130> HY140779 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 695 <212> PRT <213> Burkholderia thailandensis <400> 1 Met Val Asn His Lys Thr Gly Ser Asn Met Asn Arg Arg Asp Leu Ile   1 5 10 15 Lys Phe Leu Ser Phe Ala Ala Ser Gly Thr Ala Phe Ala Gly Leu Val              20 25 30 Arg Ser Thr Leu Ser Ser Pro Ala Ala Ser Ser Ile Thr Ala Ser Pro          35 40 45 Arg Thr Leu Asp Ala Gly Ile Gly Ile Ser Ser Pro Gln Ala Val Arg      50 55 60 Ala Ala Ala Pro Val Leu Pro Gln Lys Asp Thr Ala Ala Gly Arg Ile  65 70 75 80 Ala Arg Ala Gly Phe Leu Ala Thr Gln Arg Leu Ser Tyr Ile Trp Thr                  85 90 95 Glu His Val Pro Thr Ala Ser Gly Ile Pro Leu Ala Leu Val Thr Pro             100 105 110 Gln Asp Leu Pro Thr Ile Glu Trp Leu Ile Lys Phe Ile Ala Ile Val         115 120 125 Val Gly Val Ile Glu Asn Phe Leu Gly Ser Ala Pro Ala Thr Ala Val     130 135 140 Ala Leu Trp Arg Asp Gln Phe Ala Lys Ile Arg Val Asp Leu Leu Ser 145 150 155 160 Leu Glu Asn Leu Tyr Ser Asp Leu Thr His Asp Pro Asn Leu Gln Asp                 165 170 175 Pro Val Ala Ile Ala Glue Ala Ala Ser Ile Gln Ala Ala Leu Ile Ala             180 185 190 Leu Leu Ala Asn Val Gly Val Leu Ser Lys Asp Ile Ser Ser Le Leu         195 200 205 Gly Glu Ile Val Ser Asn His Asp Thr Arg Ser Glu Glu Asn Phe Lys     210 215 220 Ala Leu Phe Ser Thr Phe Pro Leu Pro Asp Ile Ser Ala Ala Tyr Gln 225 230 235 240 Arg Asp Asp His Phe Ala Ser Leu Arg Val Ala Gly Gln Asn Pro Val                 245 250 255 Leu Ile Lys Arg Ile Ser Gly Leu Pro Ser Lys Phe Pro Leu Thr Asn             260 265 270 Ala Gln Phe Gln Gln Val Met Gly Pro Ala Asp Asn Leu Val Ser Ala         275 280 285 Ala Ala Glu Asn Arg Leu Tyr Leu Leu Asp Tyr Val Asp Asn Gly Leu     290 295 300 Leu Ala Thr Ser Arg Ala Val Ala Lys Pro Leu Thr Gly Ile Gly Tyr 305 310 315 320 Ser Tyr Ala Pro Ile Ala Leu Phe Ala Leu Pro Arg Gly Gly Ala Ser                 325 330 335 Leu Val Pro Val Ala Ile Gln Cys Asp Gln Asp Pro Ala Thr Asn Pro             340 345 350 Leu Phe Leu Pro Ala Asp Pro Ser Gln Glu Ser Ala Tyr Trp Ala Trp         355 360 365 Gln Met Ala Lys Thr Val Val Gln Cys Ala Glu Glu Asn Tyr His Glu     370 375 380 Met Phe Val His Leu Ala Arg Thr His Leu Val Thr Gly Ala Ile Cys 385 390 395 400 Val Ala Thr His Arg Asn Leu Ala Ser Thr His Pro Leu Tyr Ala Leu                 405 410 415 Leu Met Pro His Phe Glu Gly Thr Leu Tyr Ile Asn Glu Leu Ala Ala             420 425 430 Leu Thr Leu Leu Pro Leu Met Phe Ile Asp Thr         435 440 445 Pro Ile Gln Gln Thr Gln Gln Met Val Ala Ser Asp Arg Leu Ala Phe     450 455 460 Asp Phe Tyr Asp His Met Leu Pro Asn Asp Ile Glu Met Arg Gly Val 465 470 475 480 Gly Ala Ala Asn Leu Pro Asp Tyr Pro Tyr Arg Asp Asp Gly Leu Leu                 485 490 495 Ile Trp Asn Ale Ile Ala Glu Trp Ala Lys Ala Tyr Val Asp Val Tyr             500 505 510 Tyr Lys Ser Asp Gln Asp Val Val Asp Asp Tyr Glu Leu Arg Ser Trp         515 520 525 Ala Ala Asp Ile Ile Ala Asn Gly Lys Val Lys Gly Phe Arg Pro Val     530 535 540 Arg Ser Lys Ala Gln Leu Ile Asp Val Leu Thr Met Ile Ile Phe Thr 545 550 555 560 Ala Ser Ala Gln His Ala Ala Val Asn Phe Ser Gln Ser Asp Phe Ser                 565 570 575 Thr Tyr Ala Pro Ala Leu Ser Ala Leu Leu Ser Ala Pro Ala Pro Thr             580 585 590 Ser Ala Val Gly Lys Ser Lys Ala Asp Trp Leu Lys Met Leu Pro Pro         595 600 605 Leu Val Ser Gly Ile Glu Arg Val Ala Ile Tyr Glu Ile Leu Ala Gly     610 615 620 Val Gln His Ser Ala Leu Gly Gln Tyr Arg Ser Asn Val Phe Pro Tyr 625 630 635 640 Arg Pro Leu Ile Thr Asp Pro Ala Ile Thr Gly Ser Asn Gly Pro Leu                 645 650 655 Glu His Phe Arg Gln Ala Leu Gly Asp Val Glu Ser Gln Ile Asn Ala             660 665 670 Arg Asn Ser Ile Arg Lys Thr Pro Tyr Glu Tyr Leu Leu Pro Ser Arg         675 680 685 Ile Pro Ala Ser Thr Asn Ile     690 695 <210> 2 <211> 2088 <212> DNA <213> Burkholderia thailandensis <400> 2 atggtcaatc acaaaaccgg gtcaaatatg aaccgaaggg atttaattaa attcttgagc 60 ttcgccgcca gcggaaccgc gtttgcgggg ctcgtcaggt cgactctgtc gtcaccggcc 120 gcgtcgtcga tcaccgcaag ccctcgcacg ctcgacgccg gcatcggcat ttcgtcgccg 180 caggcggttc gcgcagccgc gccggtgctg ccgcaaaaag acacggccgc aggcaggatc 240 gcacgggcgg gttttctcgc cacgcaaaga ctgagttaca tctggacgga acatgtgccg 300 accgcgagcg gcattccact tgcgctggtc acgccgcaag atcttccaac cattgaatgg 360 ctgatcaaat tcatcgcgat cgttgtgggc gtcatcgaga attttctcgg ctccgcaccc 420 gcgacggcgg ttgccctctg gcgcgatcag ttcgccaaga tcagggtgga tctgctttcc 480 ctcgaaaacc tgtattcgga tctcacccac gatccgaact tgcaagaccc ggttgccatt 540 gctcaggcag cgagcattca ggcggccttg atcgcgctgt tggcgaacgt cggcgtcttg 600 tcgaaggaca tcatttcaag attgggcgaa atcgtctcca accacgacac tcgaagcgaa 660 gagaacttca aggcgctgtt ttccaccttc ccgcttcccg acatctccgc cgcgtaccag 720 cgggacgatc atttcgcgtc tctccgggtt gccggccaga atccggtgtt gatcaagcgc 780 atctccggct tgccgtcgaa gttcccactg acgaacgctc aattccagca agtcatgggg 840 cccgccgaca atctcgtcag cgccgccgcg gagaatcggc tgtatctcct cgattacgtc 900 gacaacggtc tgttggcgac gtcgcgggcc gtggccaaac cactcacggg catcggctac 960 tcctatgcgc ccatcgcact tttcgcgctg ccgaggggcg gcgcgtccct cgttcccgtg 1020 gcgattcaat gcgatcagga tcccgcgacg aatcccctgt tcctccctgc cgatcccagt 1080 cagataatccg cctattgggc gtggcaaatg gccaagacgg tcgttcaatg cgcggaggaa 1140 aactatcacg agatgtttgt tcatctcgcg cgaacgcacc tggtgaccgg cgcgatttgc 1200 gtcgccactc accggaatct cgcgtcgacg catccgctct acgcgcttct gatgccgcac 1260 ttcgagggca ctttgtacat caacgaactc gccgcgctca cgctgcttcc accgttgatg 1320 ttcatcgata cgctgtttgc cgcgcccatc cagcagacgc aacagatggt cgcaagcgat 1380 cggctcgcgt tcgatttcta cgatcacatg ttgcccaacg acatcgaaat gcgcggagtc 1440 ggcgcggcca acctgccgga ctatccgtat cgcgacgacg gcctcctgat ttggaacgcc 1500 atcgccgaat gggcgaaggc gtatgtcgat gtctattaca agtccgatca ggatgtcgtc 1560 gatgactacg agctcaggtc ctgggccgcc gacatcatcg ccaacggcaa ggtcaaggga 1620 ttccggccgg tgcgttcgaa ggcgcaattg atcgacgtgc tgaccatgat catctttacc 1680 gcaagcgccc agcacgccgc cgtcaacttc tcgcagtcgg atttttcgac ctacgcgccc 1740 gcgctttccg cactgctgtc cgcgccggcc ccgacaagcg ccgtgggaaa aagcaaggcc 1800 gattggctga agatgctccc tcccctcgtt tcagggatcg agcgggttgc gatctacgag 1860 atcttggcgg gcgtccagca cagcgcgttg ggccagtatc gcagcaacgt atttccatat 1920 cggccgctca tcacggaccc cgcgatcacc ggaagcaacg ggccgctcga gcatttccga 1980 caggcgctcg gcgacgtcga atcgcagatc aacgctcgca acagcatacg caagacgcct 2040 tatgaatatc tgctgccgag ccggattccg gcaagcacga acatttga 2088 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 gctagcatgg tcaatcacaa aaccgg 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 aagctttcaa atgttcgtgc ttgccg 26

Claims (16)

A composition for the production of 13-hydroxylinoleic acid comprising lipoxygenase consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. delete The composition of claim 1, wherein the composition further comprises linoleic acid as a substrate. A composition for the production of 13-hydroxylinoleic acid comprising as an active ingredient a gene consisting of the nucleotide sequence of SEQ ID NO: 2 encoding a lipid oxidation enzyme. delete A method for producing 13-hydroxylinoleic acid by biotransformation by reacting an enzyme comprising an amino acid sequence of SEQ ID NO: 1, which produces a hydroxylated fatty acid from an unsaturated fatty acid, with a substrate. The method according to claim 6,
The enzyme that produces the hydroxylated fatty acid from the unsaturated fatty acid is called Burkholderia &lt; RTI ID = 0.0 &gt; thailandensis) characterized in that the origin. delete The method according to claim 6,
Wherein the substrate is linoleic acid. The method according to claim 6,
Characterized in that linoleic acid is used in a range from 5 g / l to 25 g / l in a substrate. The method according to claim 6,
Characterized in that methanol is added to the organic solvent. The method according to claim 6,
Characterized in that the reaction is carried out in the pH range of 7.0 to 8.0. The method according to claim 6,
Characterized in that the reaction is carried out at a temperature in the range of from 20 캜 to 35 캜. The method according to claim 6,
Characterized in that the concentration of the lipid oxidizing enzyme which is an enzyme which produces the hydroxylated fatty acid from the unsaturated fatty acid is treated in the range of 1.5 g / l to 3.0 g / l.
delete delete

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