WO2008133398A1

WO2008133398A1 - Method for preparing hydroxylinoleic acid using lipoxygenase from zea mays and the hydroxylinoleic acid prepared by the same

Info

Publication number: WO2008133398A1
Application number: PCT/KR2008/001049
Authority: WO
Inventors: Ok Soo Han; Sung Kuk Jang; Kyoung Won Cho; Keum Hwa Kim; Eun Ji Um
Original assignee: Industry Foundation of Chonnam National University
Current assignee: Industry Foundation of Chonnam National University
Priority date: 2007-04-30
Filing date: 2008-02-22
Publication date: 2008-11-06
Anticipated expiration: 2009-10-30

Abstract

Provided is a method for preparing hydroxylinoleic acid using maize (Zea mays) lipoxygenase and hydroxylinoleic acid prepared by the same. The method of the present invention comprises subjecting linoleic acid to dual regiospecific hydroperoxidation using a mixed solution containing linoleic acid and Zea mays lipoxygenase; reducing the resulting reaction product using triphenylphosphine or NaBH4 as a reducing agent; and separating the reduction product. The present invention enables production of hydroxylinoleic acid with controlled distribution of various hydroperoxylinoleic acids from linoleic acid, via the use of maize lipoxygenase which concurrently catalyzes 9-hydroperoxidation and 13-hydroperoxidation. Therefore, it is possible to provide various choices of biosynthetic pathways that can deal actively with a variety of stress applied to plants.

Description

[DESCRIPTION] [Invention Title]

Method for preparing hydroxylinoleic acid using lipoxygenase from Zea mays and the hydroxylinoleic acid prepared by the same.

[Technical Field]

The present invention relates to a method for preparing hydroxylinoleic acid using maize (Zea mays) lipoxygenase and hydroxylinoleic acid prepared by the same.

More specifically, the present invention relates to a method for preparing hydroxylinoleic acid with controlled distribution of various hydroperoxylinoleic acids from linoleic acid, using maize (Zea mays) lipoxygenase, and hydroxylinoleic acid prepared by the same.

[Background Art]

Lipoxygenase (LOX) is a non-heme iron dioxygenase that catalyzes hydroperoxidation of unsaturated fatty acid substrates having a cis, cw-l,4-pentadiene structure. Even though most of prokaryotes including yeast do not harbor a lipoxygenase gene, lipoxygenase is expressed at a high level in animal and plant cells and plays an important role in signal transduction pathways which are mediated by unsaturated fatty acids (Watanabe et al., 1997). Linoleic acid or linolenic acid which is used as a substrate for lipoxygenase is the major unsaturated fatty acid constituting plant membranes, and the membrane fluidity is determined by the content of these unsaturated fatty acids. The membrane fluidity plays a crucial role in adaptation of plants in response to various changes and fluctuations in abiotic environmental variables such as temperature, moisture, salts, and the like and biotic environmental variables such as invasion of pathogenic bacteria, harmful insects, fungi, and the like (Horvath et al., 1983). Therefore, an intracellular concentration of lipoxygenase substrates and lipoxygenase activity are positioned at the rate-determining step of a conversion process of these unsaturated fatty acids into other physiologically active substances by the lipoxygenase pathway, thereby resulting in formation of a plant stress hormone, e.g. jasmonic acid, as well as a plant wound hormone, e.g. traumatin, and have significant effects on another formation pathway of abscisic acid via violaxanthin.

In mammals, there are known numerous lipoxygenases responsible for peroxidation at various positions of C-5, C-8, C-12 and C-15 of arachidonic acid (C20:4) as a substrate (Brash, 1999; and Kuhn et al., 1999). Among them, 5-Lox is implicated in the biosynthesis of leukotriene which plays a major role in pathogenesis of inflammatory diseases, whereas 15-Lox is primarily responsible for the biosynthesis of prostaglandin, which in turn triggers the eicosanoid pathway of animals.

In plants, there are known some lipoxygenases responsible for hyperoxidation at C-9 and C- 13 positions of linoleic acid or linolenic acid as a substrate (Gardner, 1991). Among them, 13 -LOX catalyzes an early step in the biosynthesis of dihydrojasmonic acid and jasmonic acid from linoleic acid and linolenic acid, respectively, which consequently prompts the plant octadecanoid pathway to mediate host defense mechanisms (such as disease resistance, pest resistance, and environmental resistance) against a variety of stress applied to plants (Shibata and Axelrod, 1995; and Leon et al., 1999). 13(S)-hydroperoxy fatty acid and 9(S)-hydroperoxy fatty acid which are produced by the plant lipoxygenase serve as a substrate of hydroperoxide lyase and allene oxide synthase to thereby form the lipoxygenase pathway which will produce various kinds of oxylipins. Oxylipins including jasmonic acid, traumatic acid and α-ketol are biosynthesized from 13(S)- hydroperoxylinolenic acid which is obtained from linolenic acid, whereas oxylipins including various kinds of alkenals are biosynthesized from linoleic acid. Therefore, it can be said that the substrate specificity and positional specificity (or regiospecificity) of lipoxygenase which correspond to a starting point of the lipoxygenase pathway play a key part in what kind of compound will be produced among various kinds of oxylipins having unique physiological activity. Most of lipoxygenases catalyze regioselective and stereoselective reactions, so they may be broadly classified into 9-lipoxygenase that catalyzes hydroperoxidation at the

C-9 position of a substrate and 13 -lipoxygenase that catalyzes hydroperoxidation at the C-

13 position of a substrate. Therefore, each of these lipoxygenases produces only the corresponding specific stereoisomers. Exceptionally, it was suggested that lipoxygenases found in some plants including maize lipoxygenase exhibit a broad substrate specificity without being regioselective or stereoselective, and therefore produces a variety of reaction products.

For instance, Korean Patent Application Publication No. 2001-0085101 Al discloses that the maize lipoxygenase concurrently catalyzes 9-hydroperoxidation and 13- hydroperoxidation to produce, as reaction products, 9-hydroperoxylinoleic acid and 13- hydroperoxylinoleic acid which can be used as a starting material of diverse oxylipin biosynthetic pathways of plants.

However, the aforesaid art suffers from various disadvantages. That is, it is very difficult to calculate distribution of the corresponding reaction product due to changes of individual reaction products which may occur during separation of the reaction products, i.e. hydroperoxylinoleic acids. Further, it is impossible to control distribution of the predictable reaction products. [Disclosure] [Technical Problem]

Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a method for preparing hydroxylinoleic acid with controlled distribution of various hydroperoxylinoleic acids from linoleic acid, using maize {Zea mays) lipoxygenase.

It is another object of the present invention to provide hydroxylinoleic acid prepared by the aforesaid method.

[Technical Solution]

In accordance with an aspect of the present invention, the above and other objects can be accomplished by the provision of a method for preparing hydroxylinoleic acid comprising: subjecting linoleic acid to dual regiospecific hydroperoxidation using a mixed solution containing linoleic acid and maize {Zea mays) lipoxygenase; reducing the resulting reaction product using triphenylphosphine (TPP) or sodium borohydride (NaBH₄) as a reducing agent; and separating the resulting reduction product.

The lipoxygenase may be a protein having an amino acid sequence as set forth in SEQ ID NO: 2 or a protein having a sequence homology of more than 70% to the protein having an amino acid sequence of SEQ ID NO: 2.

The reaction product may be 13-(9Z,l lE)-hydroperoxyoctadecadienoic acid, 13- (9E, 11 E)-hydroperoxyoctadecadienoic acid, 9-( 1 OE, 12Z)-hydroperoxyoctadecadienoic acid or 9-(10E,12E)-hydroperoxyoctadecadienoic acid. A concentration of linoleic acid may be in the range of 0.03 mM to 0.5 mM. A concentration of lipoxygenase may be in the range of 0.4 /Λg/mL to 28.8 μg/mL.

The reduction product may be 13-(9Z,l lE)-hydroxyoctadecadienoic acid, 13- (9E,llE)-hydroxyoctadecadienoic acid, 9-(10E,12Z)-hydroxyoctadecadienoic acid, or 9- (10E,12E)-hydroxyoctadecadienoic acid.

When the reducing agent is triphenylphosphine (TPP), 11 to 15 molecules of 13-

(9Z,l lE)-hydroxyoctadecadienoic acid, 8 to 12 molecules of 13 -(9E, HE)- hydroxyoctadecadienoic acid and 9 to 13 molecules of 9-(1OE, 12Z)- hydroxyoctadecadienoic acid may be respectively produced relative to 10 molecules of 9- (1 OE, 12E)-hydroxyoctadecadienoic acid.

When the reducing agent is NaBH₄, 1:5 to 20 molecules of 13-(9Z₅I lE)- hydroxyoctadecadienoic acid, 9 to 13 molecules of 13-(9E,l lE)-hydroxyoctadecadienoic acid and 14 to 18 molecules of 9-(10E,12Z)-hydroxyoctadecadienoic acid may be respectively produced relative to 10 molecules of 9-(10E,12E)-hydroxyoctadecadienoic acid.

The hydroperoxidation may be carried out at a pH of 6 to 8 and a temperature of 20 to 30^°C for 10 to 20 min.

When the reducing agent is TPP, the method may further comprise adding an organic solvent to elute the reaction product after completion of the hydroperoxidation. The organic solvent may be at least one selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol and any combination thereof.

Separation of the reduction product may be carried out by HPLC.

In accordance with another aspect of the present invention, there is provided hydroxylinoleic acid prepared by the aforesaid method. [Advantageous Effects]

As will be illustrated hereinafter, the present invention enables production of hydroxylinoleic acid with controlled distribution of various hydroperoxylinoleic acids from linoleic acid, via the use of maize lipoxygenase which simultaneously catalyzes 9- hydroperoxidation and 13-hydroperoxidation. Therefore, it is possible to provide various options of biosynthetic pathways that can cope effectively with a variety of stress applied to plants.

[Description of Drawings] FIG. 1 illustrates reaction products from hydroperoxidation of linoleic acid using maize lipoxygenase;

FIG. 2 illustrates SDS-PAGE patterns of lipoxygenase isolated and purified from E. coli BL21(DE3)pLysS/pRSETB/LOXl cells which expressed maize lipoxygenase and were subjected to sonic disruption; FIG. 3 illustrates the time course UV -vis spectra of a process where maize lipoxygenase produces hydroperoxylinoleic acid using linoleic acid as a substrate;

FIG. 4 illustrates Straight-Phase HPLC (SP-HPLC) analysis results for distribution of the reduction product obtained in Example 4 (A) and distribution of the reduction product obtained in Example 5 (B); FIG. 5 illustrates the mass analysis spectra of trimethylsilylated 13 -(9Z, HE)- hydroxyoctadecadienoic acid prepared in Example 4; and

FIG. 6 illustrates the mass analysis spectra of trimethylsilylated 9-(1OE, 12Z)- hydroxyoctadecadienoic acid prepared in Example 4. [Best Mode]

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings, if necessary.

The present invention enables production of hydroxylinoleic acid with controlled distribution of various hydroperoxylinoleic acids by isolating maize lipoxygenase capable of simultaneously catalyzing 9-hydroperoxidation and 13-hydroperoxidation, preparing various hydroperoxylinoleic acids from linoleic acid using the maize lipoxygenase, reducing the resulting reaction products with a reducing agent, and then confirming distribution of the reduction products to thereby achieve controlled distribution of the reaction intermediates, hydroperoxylinoleic acids. Therefore, the present invention provides a basis which is capable of adopting diverse biosynthetic pathways that can cope actively with a variety of stress imposed on plants.

By controlling concentrations of linoleic acid and maize lipoxygenase according to the present invention, it is possible to easily modulate the distribution of various hydroperoxylinoleic acids which can be used as a starting material of diverse oxylipin biosynthetic pathways of plants. Further, the present invention is characterized in that it is possible to control a distribution ratio of hydroxylinoleic acid products depending upon kinds of reducing agents which reduces each of hydroperoxylinoleic acids.

The present invention provides a method for preparing hydroxylinoleic acid using maize lipoxygenase.

For this purpose, dual regiospecific hydroperoxidation of linoleic acid is first carried out using linoleic acid and maize lipoxygenase.

Linoleic acid (LA) used in the present invention is unsaturated fatty acid containing a cis, czs- 1,4-pentadiene unit (-CH=CH-CH₂-CH=CH-) and is used as a substrate for hydroperoxidation. The oxidation of linoleic acid into hydroperoxylinoleic acid is catalytically accelerated.

Maize (Zea mays) lipoxygenase 1 (LOXl) is used as a catalyst that facilitates to induce dual positional specific or regiospecific hydroperoxidation of linoleic acid. That is, LOXl produces a variety of reaction products via cocatalysis of 9-hydroperoxidation and 13 -hydroperoxidation.

Zea mays LOXl is a protein having a molecular weight of about 97 kD in the natural state and an amino acid sequence as set forth in SEQ ID NO: 2.

The LOXl enzyme may be a protein having an amino acid sequence as set forth in SEQ ID NO: 2 or a protein having a sequence homology of more than 70% to the protein having an amino acid sequence of SEQ ID NO: 2. That is, the lipoxygenase protein in accordance with the present invention may encompass a protein having an amino acid sequence of SEQ ID NO: 2 isolated from maize (Zea mays) and a functional equivalent thereof. As used herein, the term "functional equivalent" refers to a protein that contains additions, substitutions and/or deletions of amino acid(s) and therefore has a sequence homology of at least more than 70%, preferably more than 80%, further preferably more than 90%, and still further preferably more than 95% to the amino acid sequence of SEQ

ID NO: 2 while retaining substantially the same physiological activity as the protein having an amino acid sequence of SEQ ID NO: 2. The term "substantially the same physiological activity" refers to an activity that enhances the plant stress resistance when the gene of interest is overexpressed in plants.

The hydroperoxidation is made by mixing and reacting linoleic acid and lipoxygenase. As a solvent for mixing of linoleic acid with lipoxygenase, Tri-Cl buffer may be used. Meanwhile, the mixed solution may contain a detergent to enhance the compatibility between hydrophilic and hydrophobic groups. There is no particular limit to the detergent, as long as it is conventionally used in the art. Preferably, Tween 20

(available from Uniqema, USA) may be used which consists largely of hydrophilic groups of polyethylene glycol in admixture with hydrophobic groups of hydrocarbon.

In order to increase the catalytic activity of lipoxygenase, the hydroperoxidation may be carried out at a pH of 6 to 8 and room temperature of 20 to 30^°C for 10 to 20 min.

If the pH and temperature for hydroperoxidation are out of the range of the above- specified values, this may undesirably result in deterioration of lipoxygenase activity which makes it difficult to carry out dual positional specific hydroperoxidation of linoleic acid. Meanwhile, if a reaction time is shorter than 10 min, this may cause difficulty in completion of the reaction. On the other hand, if a reaction time is longer than 20 min, it is not desirable for process efficiency.

The hydroperoxidation product will contain a hydroperoxy (-OOH) group at the C-9 or C-13 position of linoleic acid.

FIG. 1 illustrates reaction products from hydroperoxidation of linoleic acid using maize lipoxygenase. Specifically referring to FIG. 1, lipoxygenases (13-LOX1 and 9- LOXl) concurrently catalyzes 13 -hydroperoxidation and 9-hydroperoxidation of linoleic acid (LA) to thereby result in production of 13 -(9Z₅I lE)-hydroperoxyoctadecadienoic acid (HPODE), 13-(9E,l lE)-hydroperoxyoctadecadienoic acid, 9-(1OE, 12Z)- hydroperoxyoctadecadienoic acid and 9-(10E,12E)-hydroperoxyoctadecadienoic acid.

A distribution ratio of each reaction product may be controlled by a concentration of linoleic acid used as a substrate in the present invention or a concentration of lipoxygenase that catalyzes the dual positional specific hydroperoxidation of linoleic acid. For example, a concentration of linoleic acid may be controlled in the range of 0.03 mM to 0.5 mM to modify a distribution ratio of each reaction product.

Further, a concentration of lipoxygenase may be controlled in the range of 0.4 //g/mL to 28.8 μg/mL to modify a distribution ratio of each reaction product. Next, reduction of the resulting hydroperoxidation product is carried out using triphenylphosphine (TPP) or NaBH₄ as a reducing agent.

This process results in reduction of hydroperoxylinoleic acid having -OOH into the corresponding counterpart having -OH. The present invention features use of TPP or NaBH₄ as a reducing agent for reduction of hydroperoxidation products. , First, when it is desired to use TPP for the reduction reaction, the method of the present invention may further comprise elution of the reaction products after completion of the hydroperoxidation. Elution of the reaction products may be carried out using any conventional method known in the art. For example, elution of the reaction products may be carried out using at least one organic solvent selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol and any combination thereof.

When NaBH₄ is used for the reduction reaction, it is advantageous for the process because there is no need for an additional step to elute the reaction products.

The reduction product obtained from reduction of hydroperoxidation products using TPP or NaBH₄ as a reducing agent may be 13-(9Z,l lE)-hydroxyoctadecadienoic acid (HODE), 13-(9E,l lE)-hydroxyoctadecadienoic acid, 9-(10E,12Z)- hydroxyoctadecadienoic acid or 9-(10E,12E)-hydroxyoctadecadienoic acid.

Meanwhile, the present invention is characterized in that it is possible to control an amount of respective reduction products which are produced from reduction of hydroperoxidation products using the above-mentioned reducing agent, i.e. distribution of the reduction products. When the reducing agent is TPP, 11 to 15 molecules of 13-(9Z₅I lE)- hydroxyoctadecadienoic acid, 8 to 12 molecules of 13-(9E,l lE)-hydroxyoctadecadienoic acid and 9 to 13 molecules of 9-(10E,12Z)-hydroxyoctadecadienoic acid may be respectively produced relative to 10 molecules of 9-(10E,12E)-hydroxyoctadecadienoic acid.

When the reducing agent is NaBH₄, 15 to 20 molecules of 13-(9Z₅I lE)- hydroxyoctadecadienoic acid, 9 to 13 molecules of 13-(9E,l lE)-hydroxyoctadecadienoic acid and 14 to 18 molecules of 9-(10E,12Z)-hydroxyoctadecadienoic acid may be respectively produced relative to 10 molecules of 9-(10E,12E)-hydroxyoctadecadienoic acid.

The reduction of hydroperoxidation products may be carried out at a pH of 3 to 4 for 20 to 40 min.

Then, separation of the resulting reduction product is carried out.

Separation of the reduction product may be carried out by High Performance Liquid Chromatography (HPLC). There is no particular limit to the elution buffer for HPLC, as long as it is a nonsolvent conventionally used in the art. Preferably, a mixed solvent of hexane, alcohol and acetic acid may be used.

[Mode for Invention] EXAMPLES

Now, the present invention will be described in more detail with reference to the following Examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention. Example 1

Preparation of maize lipoxygenase

E. coli strain harborin g a maize lipoxygenase gene (pX Ll /LOXl) introduced therein was cultured at 37 "C and the lipoxygenase gene (SEQ ID NO: 1, Genbank Accession No. AF271894) was then obtained using a plasmid DNA purification kit (QIAGEN Co., USA).

Polymerase Chain Reaction (PCR) was carried out using a primer pair for amplification of the gene of interest, i.e., one primer of 5'- TGCAGCTGGTC^TATGGTCG-3' (SEQ ID NO: 3) having an Ndel restriction site and another primer of 5'-AAGATTCGAATTCAGCTCAG-S' (SEQ ID NO: 4) having an EcoRl restriction site. The PCR product was introduced into a pGEM-T/Easy vector for amplification and then subcloned into NdeVEcoRl sites of a pRSETB vector to construct a recombinant LOXl expression vector, designated pRSETB/LOXl.

The recombinant vector pRSETB/LOXl was transfected into a BL21(DE3)pLysS strain which was then inoculated into an LB medium containing ampicillin (50 μg/mL) and chloramphenicol (35 mg/mL), followed by cultivation at 37^°C for 16 hours. The cells were inoculated at a dilution ratio of 1/100 in a fresh LB medium, and grown at 37^°C to an optical density of 0.6 at 600 nm and then further cultured at 25 ^°C for 12 hours after addition of an expression inducer isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 1 mM to thereby induce mass expression of the recombinant lipoxygenase

(pRSETB/LOXl).

Bacterial cells were harvested by centrifugation of the cell culture (3,000^χg, 4^°C and 15 min) and washed with 50 mM Tris-Cl buffer (pH 7.2). Then, the cells were centrifuged at 13,000*g and 4⁰C for another 1 hour and resuspended in 3 mL of Tris-Cl buffer (50 mM, pH 7.2) containing 0.1% Tween 20 and 0.2 mM protease inhibitor (phenylmethylsulfonyl fluoride (PMSF), Sigma), followed by sonic disruption to extract unpurified lipoxygenase. The obtained lipoxygenase-containing crude product was subjected to anion exchange chromatography to thereby purify lipoxygenase. For this purpose, the crude product was passed through a column (Q-sepharose resin) equilibrated with 50 rnM Tris-Cl (pH 7.2) and then Tris-Cl (50 mM, pH 7.2) containing 50 mM NaCl was passed through the column.

Example 2: Confirmation of lipoxygenase

In order to confirm isolation and purification of lipoxygenase from the sonically disrupted E. coli BL21(DE3)pLysS/pRSETB/LOXl cells, the lipoxygenase protein was separated on 10% SDS-PAGE gels and stained with Coomassie-brilliant blue. The results obtained are shown in FIG. 2.

Referring to FIG. 2, Lane M represents a molecular weight marker, Lane 1 represents an unpurified extract obtained from the E. coli strain which was disrupted by sonication, Lane 2 represents a supernatant obtained by centrifugation of the unpurified crude extract, Lane 3 represents a precipitate obtained by centrifugation of the unpurified crude extract, and Lane 4 represents lipoxygenase (97kD) purified by anion exchange chromatography. From the results of FIG. 2, it was confirmed that a staining marker of Lane 4 is lipoxygenase isolated from maize.

Example 3 : Confirmation of lipoxygenase activity

6 βg of lipoxygenase was added and reacted in 2.5 mL of Tris-Cl buffer (50 mM, pH 7.2) containing 0.5 mM linoleic acid and 0.05% Tween 20 at 25 ^°C . An amount of the reaction product produced during the lipoxygenase reaction was measured in terms of UV- Vis spectrum using a UV spectrophotometer (UV-2550) at a wavelength of 200 nm to 400 nm every 2 minutes. FIG. 3 illustrates the results of UV- Vis spectral analysis.

Referring to FIG. 3, increased absorbance at 234 nm was observed, thus confirming activity of lipoxygenase using linoleic acid as a substrate.

Example 4: Analysis of reduction products obtained from reduction of hydroperoxylinoleic acid using TPP

Hydroperoxylinoleic acid, produced from linoleic acid using maize lipoxygenase, was reduced using TPP or NaBH₄ as a reducing agent and subjected to SP-HPLC to thereby separate four different regiospecific reduction products. Then, distribution of individual reduction products was examined.

First, 72 βg of lipoxygenase obtained as above was added and reacted in 2.5 mL of Tris-Cl buffer (50 mM, pH 7.2) containing 0.5 mM linoleic acid and 0.05% Tween 20 (Uniqema) at 25 ^°C for 15 min. The resulting reaction solution was adjusted to have a pH of 4 by addition of a chilled methanol/acetic acid mixture, and the pH-adjusted reaction solution was then passed through a solid-phase extraction cartridge (Sep-pak C₁₈). Next, the cartridge was washed with 10 mL of a 10% methanol solution, and then 10 mL of water. Thereafter, residual water in the cartridge was completely removed with a syringe, and 3 mL of 2-propanol was passed through the cartridge to elute the reaction product. TPP was added to the eluate, followed by reaction at 0^°C for 30 min. Using a rotary vacuum evaporator, 2-propanol was then removed from the reaction solution to obtain a reduction product. Next, the reduction product was mixed in an SP-HPLC solvent (n-hexane/2- propanol/acetic acid = 100/2/0.1, v/v/v) and the resulting solution was loaded on an SP- HPLC column, followed by fractionation of the reduction product. Four fractionated reduction products obtained were designated I, II, III, and IV, respectively. FIG. 4A illustrates SP-HPLC analysis results for distribution of individual reduction products.

Mass analysis of four reduction products was carried out and the structure of the products was identified. Four fractionated reduction products were dried under vacuum and

10 μJL of SIGMA-SIL-A was added thereto, followed by reaction at 80 ^°C for 5 min. Mass analysis for 1 μi of each reaction solution was carried out by GC-MS. For this purpose, a

GC column (HP-5MS) with a size of 30 m x 0.25 mm x 0.25 μm was used. Injector and detector temperatures were set at 260 ^°C and 300 °C , respectively. The column temperature was 100 ^°C to 160 ^°C (20 ^°C /min), and 260 ^°C to 280 ^°C (4 ^°C /min).

The mass analysis results showed that the fractionated reduction products I and II have identical mass patterns. FIG. 5 illustrates mass analysis spectra of trimethylsilylated 13-(9Z,l lE)-hydroxyoctadecadienoic acid (HODE) corresponding to product I. Further, products III and IV had identical mass patterns. FIG. 6 illustrates mass analysis spectra of trimethylsilylated 9-(10E,12Z)-hydroxyoctadecadieαoic acid (HODE) corresponding to product III.

For structural analysis of individual reduction products which were not resolvable by the GC-MS analysis, ¹H NMR analysis was carried out. Each of four reduction products I, II, III and IV which were fractionated by SP-HPLC was dried in vacuo, and was dissolved in 0.45 mL of a CDCl₃ solution containing 0.03% trimethylsilane (v/v). ¹H NMR data at 500 MHz was obtained using a Bruker Avance 500 spectrometer. The results obtained are given in Table 1 below.

Table 1

Referring to FIGS. 4, 5 and 6 in conjunction with Table 1, it was confirmed that four reduction products prepared in Example 4 correspond to I: 13-(9Z₅I lE)-HODE, II: 13-(9E₅I lE)-HODE, III: 9-(1OE, 12Z)-HODE, and IV: 9-(10E₅12E)-HODE₅ respectively.

Example 5: Analysis of reduction products obtained from reduction of hydroperoxylinoleic acid using NaBH₄

72 μg of lipoxygenase obtained as above was added and reacted in 2.5 niL of

Tris-Cl buffer (50 mM, pH 7.2) containing 0.5 mM linoleic acid and 0.05% Tween 20 (Uniqema) at 25 ^°C for 15 min. The reaction was stopped with addition Of NaBH₄. The resulting reaction solution was adjusted to have a pH of 3 by addition of IN HCl, and the pH-adjusted reaction solution was immediately passed through a solid-phase extraction cartridge (Sep-pak C₁₈) to afford a reduction product. Next, the reduction product was subjected to fractionation and mass analysis in the same manner as in Example 4. Four fractionated reduction products obtained were designated I, II, III, and IV, respectively. FIG. 4B illustrates SP-HPLC analysis results for distribution of individual reduction products.

Table 2 below shows SP-HPLC analysis results for distribution ratios of the reduction products of Examples 4 and 5 shown in FIG. 4. Referring to Table 2, it was confirmed that the reduction products produced according to Examples 4 and 5 exhibit varying distribution ratios depending upon kinds of reducing agents.

Table 2

Example 6: Effects of linoleic acid concentrations on distribution of reduction products

Reduction products were obtained in the same manner as in Example 4, except that linoleic acid used as a substrate was added at various different concentrations of 0.03, 0.04, 0.05, 0.1 and 0.5 mM. Then, the products were fractionated, followed by mass analysis.

Table 3 below shows distribution ratios of the reduction products with respect to varying concentrations of linoleic acid.

Table 3

Referring to Table 3, it was confirmed that an increase in the linoleic acid concentration leads to increased production of products I and III, and decreased production of products II and IV. From these results, it can be seen that varying concentrations of linoleic acid used as a substrate lead to changes in production/distribution of reduction products. Therefore, it can be seen that production/distribution of hydroperoxylinoleic acids corresponding to intermediate products prior to the reduction step was controlled by a concentration of the substrate linoleic acid.

Example 7: Effects of lipoxygenase concentrations on distribution of reduction products

Reduction products were obtained in the same manner as in Example 4, except that lipoxygenase was added at a concentration of 1.0 μg or 72 μg. Then, the products were fractionated, followed by mass analysis.

Table 4 below shows distribution ratios of the reduction products with respect to varying concentrations of lipoxygenase.

Table 4

Referring to Table 4, it was confirmed that an increase in lipoxygenase concentration leads to decreased distribution ratios of products I and III, and increased distribution ratios of products II and IV. From these results, it can be seen that varying concentrations of lipoxygenase used resulted in changes in production/distribution of reduction products. Therefore, it can be seen that production/distribution of hydroperoxylinoleic acids corresponding to intermediate products prior to the reduction step was controlled by a content of lipoxygenase.

[Industrial Applicability]

As apparent from the above description, the present invention enables control of distribution of various hydroperoxylinoleic acids which can be utilized as a starting material of diverse oxylipin biosynthetic pathways of plants. Therefore, the present invention provides a basis which allows for broad options of biosynthetic pathways that can cope effectively with a variety of stress applied to plants.

Claims

[CLAIMS] [Claim 1 ] A method for preparing hydroxylinoleic acid comprising: subjecting linoleic acid to dual regiospecific hydroperoxidation using a mixed solution containing linoleic acid and maize (Zea mays) lipoxygenase; reducing the resulting reaction product using triphenylphosphine (TPP) or sodium borohydride (NaBH₄) as a reducing agent; and separating the resulting reduction product.

[Claim 2] The method according to claim 1, wherein the lipoxygenase is a protein having an amino acid sequence as set forth in SEQ ID NO: 2 or a protein having a sequence homology of more than 70% to the protein having an amino acid sequence of SEQ ID NO: 2.

[Claim 3] The method according to claim 1 , wherein the reaction product is 13- (9Z, 11 E)-hydroperoxyoctadecadienoic acid, 13 -(9E, 11 E)-hydroperoxyoctadecadienoic acid, 9-(10E,12Z)-hydroperoxyoctadecadienoic acid, or 9-(1OE, 12E)- hydroperoxyoctadecadienoic acid.

[Claim 4] The method according to claim 1, wherein a concentration of linoleic acid is in the range of 0.03 mM to 0.5 mM.

[Claim 5] The method according to claim 1, wherein a concentration of lipoxygenase is in the range of 0.4 //g/mL to 28.8 //g/mL.

[Claim 6] The method according to claim 1, wherein the reduction product is 13- (9Z,l lE)-hydroxyoctadecadienoic acid, 13-(9E,l lE)-hydroxyoctadecadienoic acid, 9- (10E,12Z)-hydroxyoctadecadienoic acid or 9-(10E,12E)-hydroxyoctadecadienoic acid.

[Claim 7] The method according to claim 6, wherein 11 to 15 molecules of 13-

(9Z,l lE)-hydroxyoctadecadienoic acid, 8 to 12 molecules of 13-(9E₅I lE)- hydroxyoctadecadienoic acid and 9 to 13 molecules of 9-(1OE, 12Z)- hydroxyoctadecadienoic acid are respectively produced relative to 10 molecules of 9- (10E,12E)-hydroxyoctadecadienoic acid, when the reducing agent is TPP.

[Claim 8] The method according to claim 6, wherein 15 to 20 molecules of 13- (9Z,l lE)-hydroxyoctadecadienoic acid, 9 to 13 molecules of 13-(9E₅I lE)- hydroxyoctadecadienoic acid and 14 to 18 molecules of 9-(1OE, 12Z)- hydroxyoctadecadienoic acid are respectively produced relative to 10 molecules of 9- (10E,12E)-hydroxyoctadecadienoic acid, when the reducing agent is NaBH₄.

[Claim 9] The method according to claim 1, wherein the hydroperoxidation is carried out at a pH of 6 to 8 and a temperature of 20 to 30^°C for 10 to 20 min.

[Claim 10] The method according to claim 1, further comprising adding an organic solvent to elute the reaction product after completion of the hydroperoxidation, when the reducing agent is TPP.

[Claim 11 ] The method according to claim 10, wherein the organic solvent is at least one selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol and any combination thereof.

[Claim 12] The method according to claim 1, wherein the separation of the reduction product is carried out by HPLC.

[Claim 13] Hydroxylinoleic acid which is prepared by the method of any one of claims 1 to 12.