KR100938714B1 - Method for preparing hydroxylinoleic acid using lipoxygenase from Zea mays and the hydroxylinoleic acid prepared by the same - Google Patents

Abstract

Provided are a method for preparing hydroxylinoleic acid using a corn-derived lipoxygenase enzyme, and hydroxylinoleic acid produced thereby.

Hydroxylinoleic acid production method using a corn-derived lipoxygenase enzyme according to the present invention is a bi-specific hydroperoxidation reaction against linoleic acid using a mixed solution containing linoleic acid and corn-derived lipoxygenase enzyme Performing; Reducing the reaction product using triphenylphosphine or NaBH ₄ as a reducing agent; And separating the reduction product.

According to the present invention, plants can be produced by controlling the distribution of various hydroperoxylinoleic acid from linoleic acid using a lipoxygenase enzyme derived from corn which simultaneously catalyzes 9-hydroperoxidation and 13-hydroperoxidation. It has the effect of choosing different biosynthetic pathways that can cope with a wide range of stresses.

Description

Method for preparing hydroxylinoleic acid using corn-derived lipoxygenase enzyme and hydroxylinoleic acid prepared by the same {Method for preparing hydroxylinoleic acid using lipoxygenase from Zea mays and the hydroxylinoleic acid prepared by the same}

The present invention relates to a method for preparing hydroxylinoleic acid using a corn-derived lipoxygenase enzyme, and to hydroxylinoleic acid produced by the present invention, and more particularly, to various hydropers from linoleic acid using a corn-derived lipoxygenase enzyme. The present invention relates to a method for preparing hydroxylinoleic acid which can be produced by adjusting the distribution of oxylinoleic acid, and to hydroxylinoleic acid produced thereby.

Lipoxigenase is an iron dioxide containing no heme group and catalyzes the hydroperoxidation reaction of unsaturated fatty acid substrates with cis , cis -1,4-pentadiene structures. Although not present in most prokaryotic organisms such as yeast, they are expressed at high levels in animals and plants and play an important role in signaling mediated by unsaturated fatty acids (Watanabet al., 1997).

Linoleic acid and linolenic acid, which are used as substrates of lipoxygenase, are the main unsaturated fatty acids that make up the membrane of a plant. The fluidity of the membrane is determined by the content of these unsaturated fatty acids. Biological changes such as invasion of pathogens, pests and fungi play an important role in the adaptation of plants (Horvath et al., 1983). Therefore, the concentration of intracellular lipoxygenase substrate and the activity of lipoxygenase are located at the rate determining stage in the process of converting these unsaturated fatty acids into other bioactive substances by the lipoxygenase pathway. In addition to acid, it forms traumatin, a wound hormone, and violaxanthin, which significantly affects the formation of another abscisic acid.

In mammals, lipoxygenase which peroxidates at various positions such as C-5, C-8, C-12, C-15 using arachidonic acid (C20: 4) as a substrate, Known (Brash, 1999; Kuhnet al., 1999), among which 5-Lox biosynthesizes leukotriene, which plays an important role in inflammatory diseases, and 15-Lox is a major step in the biosynthesis process of prostaglandin. It is responsible for triggering the animal's eicosanoid route.

In plants, lipoxygenase is known to hyperoxidate at positions C-9 and C-13 using linoleic acid or linolenic acid as a substrate (Gardner, 1991). Among these, 13-lipoxygenase is derived from linoleic acid and linolenic acid. By catalyzing the early stages leading to the biosynthesis of dihydrojasmonic acid and jasmonic acid, respectively, they trigger the octadecanoid pathways of plants, adding to disease resistance, pest resistance, and environmental resistance. Loss is an important enzyme that mediates defense mechanisms against various types of stress (Shibata and Axelrod, 1995; Leon et al., 1999). The 13 (S) -hydroperoxy fatty acid and 9 (S) -hydroperoxy fatty acid produced by the lipoxygenase of plants are characterized by hydroperoxide lyase and allene oxide synthase. It forms a lipoxygenase pathway that acts as a substrate to produce various kinds of oxylipin compounds. Oxylipin compounds such as jasmonic acid, traumatic acid and α-ketol are obtained from linolenic acid 13 (S). -Biosynthesized from hydroperoxy linolenic acid, various types of oxylipin compounds such as alkenals are biosynthesized from linoleic acid. Therefore, the substrate specificity and the reaction specificity of lipoxygenase, which is the starting point of the lipoxygenase pathway, play a major role in the generation of oxylipin compounds among the various kinds of oxylipin compounds each having unique physiological activities. can do.

On the other hand, since most lipoxygenases catalyze regioselective and stereoselective reactions, hydroperoxidation at 9-lipoxygenase and C-13 positions catalyzes hydroperoxidation at the C-9 position of the substrate. It is divided into 13-lipoxygenase which catalyzes, and each of the lipoxygenases produces only specific stereoisomers.

Exceptionally, it has been suggested that lipoxygenases found in some plants, such as maize, are not regioselective or stereoselective and exhibit broad substrate specific properties, producing a variety of reaction products.

For example, in Korea Patent Publication No. 2001-0085101, corn-derived lipoxygenase simultaneously catalyzes 9-hydroperoxidation and 13-hydroperoxidation as reaction products, and various oxylipin biosynthesis present in plants. It has been disclosed that it is possible to produce 9-hydroperoxylinoleic acid and 13-hydroperoxylinoleic acid which can be used as starting materials for the route.

 However, it is very difficult to estimate the distribution of each reaction product due to the changes of each reaction product occurring during the separation of the hydroperoxylinoleic acid reaction product produced above, and furthermore, to control the distribution of the predictable reaction product. There was no problem.

Accordingly, the first problem to be solved by the present invention is to provide a method for preparing hydroxylinoleic acid that can be produced by adjusting the distribution of various hydroperoxylinoleic acid from linoleic acid using a corn-derived lipoxygenase enzyme.

In addition, a second problem to be solved by the present invention is to provide a hydroxylinoleic acid prepared by the method for preparing hydroxylinoleic acid.

The present invention to solve the first problem,

Performing a bi-specific hydroperoxidation reaction on linoleic acid using a mixed solution comprising linoleic acid and corn-derived lipoxygenase enzyme; the reaction product is triphenylphosphine (TPP) or NaBH ₄ as a reducing agent. Reducing using; And it provides a method for producing hydroxylinoleic acid comprising the step of separating the reduction product.

In addition, the lipoxygenase enzyme may be a protein of SEQ ID NO: 2 or a protein having a sequence homology of 70% or more with the protein of SEQ ID NO: 2.

On the other hand, the reaction product is 13- (9Z, 11E) -hydroperoxyoctadecadenoic acid, 13- (9E, 11E) -hydroperoxyoctadecadenoic acid, 9- (10E, 12Z) -hydroperoxyocta Decadienic acid or 9- (10E, 12E) -hydroperoxyoctadecadienoic acid.

In addition, the concentration of linoleic acid may be 0.03mM to 0.5mM.

In addition, the concentration of the lipoxygenase enzyme may be 0.4 μg / ml to 28.8 μg / ml.

Meanwhile, the reduction product is 13- (9Z, 11E) -hydroxyoctadecadienoic acid, 13- (9E, 11E) -hydroxyoctadecadienoic acid, 9- (10E, 12Z) -hydroxyoctadecadienoic acid Or 9- (10E, 12E) -hydroxyoctadecadienoic acid.

In addition, when the reducing agent is TPP, 11 to 15 molecules of the 13- (9Z, 11E) -hydroxyoctadecadiic acid relative to the 10 molecules of 9- (10E, 12E) -hydroxyoctadecadienoic acid, 13 8-12 molecules of-(9E, 11E) -hydroxyoctadecadienoic acid and 9-13 molecules of 9- (10E, 12Z) -hydroxyoctadecadienoic acid may be produced.

In addition, when the reducing agent is NaBH ₄ , 15 to 20 molecules of the 13- (9Z, 11E) -hydroxyoctadecadienoic acid with respect to the 10-molecule of 9- (10E, 12E) -hydroxyoctadecadienoic acid, the 9-13 molecules of 13- (9E, 11E) -hydroxyoctadecadienoic acid and 14-18 molecules of said 9- (10E, 12Z) -hydroxyoctadecadienoic acid can be produced.

In addition, the hydroperoxidation reaction may be performed for 10 to 20 minutes at pH 6-8 and temperature 20-30 ℃.

On the other hand, when the reducing agent is TPP, after the completion of the hydroperoxidation reaction, may further comprise the step of eluting the reaction product by adding an organic solvent.

In addition, the organic solvent may be at least one selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol and mixtures thereof.

In addition, the separating of the reduction product may be performed by HPLC.

The present invention to solve the second problem,

Provided is hydroxylinoleic acid prepared by the method for preparing hydroxylinoleic acid.

According to the present invention, plants can be produced by controlling the distribution of various hydroperoxylinoleic acid from linoleic acid using a lipoxygenase enzyme derived from corn which simultaneously catalyzes 9-hydroperoxidation and 13-hydroperoxidation. It has the effect of choosing different biosynthetic pathways that can cope with a wide range of stresses.

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

The present invention isolates lipoxygenase enzymes derived from corn which can catalyze both 9-hydroperoxidation and 13-hydroperoxidation at the same time, and the various hydroperoxylinoleic acid from linoleic acid using the lipoxygenase enzyme. By confirming the distribution of the reduction product obtained by reducing it with a reducing agent, it is possible to implement a control of the distribution of various hydroperoxylinoleic acid products, which are reaction intermediate products, to cope with a wide range of stresses on plants It provides the basis for choosing biosynthetic pathways.

The present invention can easily control the distribution of various hydroperoxylinoleic acid products that can be used as starting materials for various oxylipin biosynthetic pathways present in plants by adjusting the linoleic acid concentration and the concentration of corn lipoxygenase enzyme. In addition, the distribution ratio of the hydroxylinoleic acid product can be adjusted according to the reducing agent for reducing each of the hydroperoxylinoleic acid.

The present invention provides a method for preparing hydroxylinoleic acid using a corn-derived lipoxygenase enzyme.

First, a biposition specific hydroperoxidation reaction is carried out on linoleic acid using linoleic acid and maize-derived lipoxygenase enzymes.

Linoleic acid (LA) used in the present invention is an unsaturated fatty acid of cis , cis- 1,4-pentadiene structure (-CH = CH-CH ₂ -CH = CH-), and is a substrate of a hydroperoxidation reaction. Used as Linoleic acid is catalyzed by an oxidation reaction to convert from linoleic acid to hydroperoxylinoleic acid.

Maize derived lipoxygenase (LOX1) enzyme is used as a catalyst to promote bilocation specific hydroperoxidation to linoleic acid. That is, the lipoxygenase enzyme simultaneously catalyzes 9-hydroperoxidation and 13-hydroperoxidation to produce various reaction products.

Corn derived lipoxygenase enzyme is a protein of SEQ ID NO: 2 having a molecular weight of about 97 kD in its natural state.

In addition, the lipoxygenase enzyme may be a protein having a sequence homology of 70% or more with the protein of SEQ ID NO: 2 or the protein of SEQ ID NO: 2. That is, the range of the lipoxygenase protein according to the present invention includes a protein having an amino acid sequence represented by SEQ ID NO: 2 isolated from corn and a functional equivalent of the protein. The 'functional equivalent' means at least 70%, preferably 80%, more preferably 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution or deletion of amino acids. It refers to a protein having 95% or more of sequence homology and showing substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2. Here, "substantially homogeneous physiological activity" means activity that increases the stress resistance of the plant when overexpressed in the plant.

The hydroperoxidation is accomplished by mixing the linoleic acid and the lipoxygenase enzyme to react. At this time, as a mixed solvent for mixing the linoleic acid and lipoxygenase enzyme, Tri-Cl buffer (buffer) can be used.

On the other hand, the mixed solution may include a surfactant (detergent) in order to increase the compatibility between the hydrophilic group and the hydrophobic group, any surfactants that are commonly used can be used without limitation, preferably, mainly hydrophilic group of polyethylene glycol Tween 20 (trade name of Uniqema, USA) containing a mixture of hydrophobic groups and hydrocarbons can be used.

In order to increase the catalytic activity of the lipoxygenase enzyme, the hydroperoxidation reaction may be performed at a pH of 6 to 8 and room temperature, that is, at a temperature of 20 to 30 ° C. for 10 to 20 minutes.

In this case, when the pH range and the temperature range are out of range, the activity of the lipoxygenase enzyme is lowered, so that bi-specific hydroperoxidation to linoleic acid is difficult to be performed, and the reaction time is less than 10 minutes. When the reaction is difficult to terminate and exceeds 20 minutes, the reaction efficiency is not preferable.

The product obtained through the hydroperoxidation reaction has a hydroperoxy group (hydroperoxy, -OOH) on the carbon at position 9 or 13 of linoleic acid.

Specifically, referring to Figure 1 showing the reaction product of the hydroperoxidation reaction of linoleic acid using a corn-derived lipoxygenase enzyme, the lipoxygenase enzymes (13-LOX1 and 9-LOX1) is linoleic acid (LA 13- (9Z, 11E) -hydroperoxyoctadecadienoic acid (HPODE), 13- (9E, 11E)-by simultaneously catalyzing 13-hydroperoxidation and 9-hydroperoxidation for Hydroperoxyoctadecadienoic acid, 9- (10E, 12Z) -hydroperoxyoctadecadienoic acid and 9- (10E, 12E) -hydroperoxyoctadecadienoic acid are produced.

The distribution ratio of each of the reaction products can be controlled by the concentration of linoleic acid used as a substrate in the present invention or by the concentration of lipoxygenase enzyme catalyzing biposition specific hydroperoxidation for linoleic acid.

For example, the distribution ratio of each of the reaction products may be adjusted by adjusting the concentration of linoleic acid to 0.03 mM to 0.5 mM.

Similarly, by adjusting the concentration of the lipoxygenase enzyme from 0.4 μg / ml to 28.8 μg / ml, the distribution rate of each of the reaction products may be adjusted.

Next, a step of reducing the reaction product generated by the hydroperoxidation reaction using triphenylphosphine (TPP, hereinafter referred to as TPP) or NaBH ₄ which is a reducing agent is performed.

This process is a reduction reaction in which the hydroperoxylinoleic acid having -OOH has -OH, and according to the present invention, it is characterized in that using TPP or NaBH ₄ as a reducing agent for the reduction reaction.

First, when using TPP for the reduction reaction may further comprise eluting the reaction product after performing the hydroperoxidation reaction. The elution method may use a general method known in the art. For example, the reaction product may be eluted using at least one organic solvent selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol and mixtures thereof.

On the other hand, in the case of using NaBH ₄ for the reduction reaction there is no need for the step of eluting the reaction product separately there is an advantage in the process.

The reduction product obtained from the reduction reaction using the reducing agent TPP or NaBH ₄ is 13- (9Z, 11E) -hydroxyoctadecadienoic acid (HODE), 13- (9E, 11E) -hydroxyoctadecadiene Acid, 9- (10E, 12Z) -hydroxyoctadecadienoic acid or 9- (10E, 12E) -hydroxyoctadecadienoic acid.

On the other hand, the present invention is characterized in that according to the reduction method according to the reducing agent, the amount of each of the reduction products generated afterwards, that is, the distribution of each reduction product can be adjusted.

For example, when the reducing agent is TPP, 11 to 15 molecules of the 13- (9Z, 11E) -hydroxyoctadecadienoic acid relative to the 10 molecules of 9- (10E, 12E) -hydroxyoctadecadienoic acid, 8 to 12 molecules of the 13- (9E, 11E) -hydroxyoctadecadienoic acid and 9 to 13 molecules of the 9- (10E, 12Z) -hydroxyoctadecadienoic acid may be generated.

In addition, when the reducing agent is NaBH ₄ , 15 to 20 molecules of the 13- (9Z, 11E) -hydroxyoctadecadienoic acid with respect to the 10-molecule of 9- (10E, 12E) -hydroxyoctadecadienoic acid, the 9-13 molecules of 13- (9E, 11E) -hydroxyoctadecadienoic acid and 14-18 molecules of said 9- (10E, 12Z) -hydroxyoctadecadienoic acid can be produced.

On the other hand, the reduction reaction may be performed for 20 to 40 minutes at pH 3-4.

Next, the step of separating the reduction product generated by the reduction reaction is performed.

Separation of the reduction product may be performed by High Performance Liquid Chromatography (HPLC), and may be used without limitation as long as it is a non-solvent commonly used as an elution buffer for HPLC. The mixed solvent of hexane, alcohol, and acetic acid can be used.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments, but the present invention is not limited thereto.

Example 1

Corn Origin Lipoxygenase  Preparation of the enzyme

Escherichia coli (E. coli) into which the corn lipoxygenase gene (pXL1 / LOX1) was introduced was incubated at 37 ° C, and then ligated using a plasmid DNA purification kit (QIAGEN Co., USA). The foxygenase gene (SEQ ID NO 1: Genbank Accession No. AF271894) was obtained.

A primer having a Nde I site, which is a primer pair for amplification of the gene, obtained as 5'-TGCAGCTGGT CATATG GTCG-3 '(SEQ ID NO: 3) and a primer having an EcoR I site, 5'-AAGATTC GAATTC AGCTCAG-3' ( After performing the polymerase chain reaction using the SEQ ID NO: 4) (Polymerase Chain Reaction (hereinafter, referred to as PCR)), the fragment obtained by the PCR was introduced into the pGEM-T / Easy vector and then amplified, and then pRSETB The recombinant lipoxygenase enzyme (pRSETB / LOX1) of lipoxygenase was prepared by subcloning the Nde I site and EcoR I site of the vector.

The recombinant lipoxygenase enzyme (pRSETB / LOX1) prepared above was introduced into the BL21 (DE3) pLysS strain to transform the transformed BL21 (DE3) pLysS strain to antibiotic ampicillin (50 μg / ml) and chloramphenicol ( 35 mg / ml) was inoculated in LB medium and incubated for 16 hours at 37 ℃. Inoculate 1/100 of this in LB medium, incubate at 37 ° C until the optical density becomes 0.6 at a wavelength of 600 nm, and add IPTG, an expression inducer, to a final concentration of 1 mM, and then at 25 ° C. Incubation was performed for 12 hours to induce mass expression of the recombinant lipoxygenase enzyme (pRSETB / LOX1).

The culture cultured above was centrifuged at 3,000 × g for 15 minutes at 4 ° C. to collect strains, and then washed with 50 mM Tris-Cl (pH 7.2) buffer. The washed strains were again centrifuged at 13,000 × g for 1 hour at 4 ° C. to collect strains, which were tris-Cl buffer (50 mM) containing 0.1% Tween 20 and 0.2 mM protease inhibitor (PMSF, sigma). , pH 7.2) was dissolved in 3 ml and ultrasonically crushed to extract the lipoxygenase enzyme without purification. Crude solution containing the lipoxygenase enzyme obtained above was passed through a column (Q-sepharose resin) equilibrated with 50 mM Tris-Cl (pH 7.2) buffer and then Tris-Cl containing 50 mM NaCl. The lipoxygenase enzyme was purified via anionic ion exchange chromatography through (50 mM, pH 7.2).

Example 2

Lipoxygenase  Identification of enzyme

Stained with Coomassie-brilliant blue on a 10% SDS-PAGE gel to confirm that lipoxygenase enzyme was isolated and purified from the sonicated Escherichia coli BL21 (DE3) pLysS / pRSERB / LOX1 cells. The results are shown in FIG.

Referring to Figure 2, M is a molecular marker, line 1 is an unpurified extract obtained from ultrasonically crushed strain, line 2 is a supernatant obtained by centrifugation of the unpurified extract, line 3 is obtained by centrifugation of the unpurified extract Precipitate, line 4 shows purified lipoxygenase (97kD) by anionic ion exchange chromatography, through which it was confirmed that the staining marker of line 4 is a lipoxygenase enzyme isolated from corn.

Example 3

Lipoxygenase  Confirmation of enzyme activity

To 2.5 ml of Tris-Cl (50 mM, pH 7.2) buffer containing 0.5 mM linoleic acid and 0.05% Tween 20, 6 µg of lipoxygenase enzyme was added and reacted at 25 ° C. As the reaction proceeds, the amount of the reaction product generated is measured by UV spectrophotometer (UV-2550) once every two minutes in the range of 200nm to 400nm, UV-Vis spectrum measurement results are shown in Figure 3 Shown in

Referring to FIG. 3, the absorbance increased at 234 nm to confirm the activity of the lipoxygenase enzyme based on linoleic acid.

Example 4

TPP  Analysis of reduction result by using reduction

Hydroperoxylinoleic acid produced from linoleic acid was reduced using corn-derived lipoxygenase enzyme using TPP or NaBH ₄ as a reducing agent, followed by SP-HPLC to isolate four site-specific reduction products. The distribution of the result was observed.

First, 72 μl of the lipoxygenase enzyme obtained above was added to 2.5 ml of Tris-Cl buffer solution (50 mM, pH 7.2) containing 0.5 mM linoleic acid and 0.05% of Tween 20 (manufactured by Uniqema), and the mixture was reacted at 25 ° C. for 15 minutes. After adjusting the pH to 4 by adding a mixed solution of cooled methanol and acetic acid to the reaction solution, the pH-adjusted reaction solution was passed through a solid-phase extraction cartridge (Sep-pak C ₁₈ ). The cartridge was then washed with 10 ml of 10% methanol solution followed by 10 ml of water. Thereafter, water remaining in the cartridge was completely removed by using a syringe, and then the reaction product was eluted by passing 3 ml of 2-propanol, and TPP was added to the eluate and reacted at 0 ° C. for 30 minutes. Next, 2-propanol was removed from the reaction solution using a rotary vacuum evaporator to obtain a reduction product. Next, the reduction product was mixed with SP-HPLC solvent (n-hexane / 2-propanol / acetic acid = 100/2 / 0.1, v / v / v) and then injected into the SP-HPLC column to reduce the reduction product. Fractionated.

The fractionated four reduction products were divided into I, II, III, and IV, respectively, and the results of analyzing the distribution of each reduction product through SP-HPLC are shown in FIG. 4A.

 Then, the mass reduction of the four reduction products obtained above was carried out to identify the structure of the reduction products. Each of the four reduction products fractions were dried in vacuo, and then 10 μl of SIGMA-SIL-A was added thereto, followed by reaction at 80 ° C. for 5 minutes. At this time, GC column (HP-5MS) having a width × length × thickness of 30m × 0.25mm × 0.25㎛ is used, and the temperature of injector and detector is 260 ℃ and 300 ℃, and the column temperature conditions are 100 ℃ ~ 160 ℃ (20 ° C./min) and 260 ° C. to 280 ° C. (4 ° C./min).

As a result of the mass spectrometry, the mass patterns of I and II of the fractionated reduction products were the same, and trimethylsilyl 13- (9Z, 11E) -hydroxyoctadecadienoic acid (HODE) corresponding to I was obtained. The mass spectrometry spectrum of is as shown in Figure 5a. In addition, the mass patterns of Nos. III and IV were the same, and the mass spectrometry spectra of trimethylsilyl 9- (10E, 12Z) -hydroxyoctadecadienoic acid (HODE) corresponding to No. III were as shown in FIG. 5B. .

In addition, ¹ H NMR analysis was performed to analyze the structure of each reduction product which could not be distinguished through the GC-MS. Each of the four reduction products I, II, III, and IV fractionated through SP-HPLC were vacuum dried, dissolved in 0.45 ml of a solution of CDCl ₃ containing 0.03% trimethylsilane (v / v), and then Bruker Avance 500 spectrometer. ¹ H NMR data was obtained at 500 MHz using Table 1 below.

4, 5a, 5b and Table 1, the four reduction products prepared in Example 4 are I: 13- (9Z, 11E) -HODE, II: 13- (9E, 11E) -HODE, It can be seen that III: 9- (10E, 12Z) -HODE and IV: 9- (10E, 12E) -HODE.

Example 5

NaBH ₄ Analysis of Reduction Products by Reduction Using

To 2.5 ml of Tris-Cl buffer solution (50 mM pH 7.2) containing 0.5 mM linoleic acid and 0.05% Tween 20 (manufactured by Uniqema), 72 µg of the lipoxygenase enzyme obtained above was added and reacted at 25 ° C. for 15 minutes. The reaction was stopped by addition of NaBH ₄ to the reaction solution, pH was adjusted to 3 by addition of 1N HCl, and the reaction solution was then passed through a solid-phase extraction cartridge (Sep-pak C ₁₈ ) to reduce the pH. The resulted product was obtained, and the reduction product was fractionated in the same manner as in Example 4, followed by mass spectrometry.

The four reduction products fractionated are divided into I, II, III, and IV, respectively, and the results of analyzing the distribution of each reduction product through SP-HPLC are shown in B of FIG. 4.

On the other hand, the results of analyzing the distribution ratio of each reduction product prepared by Examples 4 and 5 shown in Figure 4 through the SP-HPLC Hereinafter, it is shown in Table 2. Referring to Table 2, the reduction product produced according to Example 4 and Example 5 is It can be seen that each distribution ratio varies depending on the reducing agent.

I 13- (9Z, 11E) -HODE II 13- (9E, 11E) -HODE III 9- (10E, 12Z) -HODE IV 9- (10E, 12E) -HODE Example 4 29.3% 23.9% 24.8% 22.0% Example 5 32.9% 20.2% 28.9% 18.0%

Example 6

Linoleic  Concentration control

A reduction product was obtained in the same manner as in Example 4 except that the concentration of linoleic acid used as the substrate was added to 0.03 mM, 0.04 mM, 0.05 mM, 0.1 mM, or 0.5 mM, and the mass fraction was analyzed. Was carried out.

The distribution rate of the reduction product according to the linoleic acid concentration is shown in Table 3 below.

LOX1 (μg) LA (mM) Ⅰ 13- (9Z, 11E) -HODE (%) III 9- (10E, 12Z) -HODE (%) Ⅰ + Ⅲ (%) II 13- (9E, 11E) -HODE (%) IV 9- (10E, 12E) -HODE (%) Ⅱ + IV (%) 72 0.03 19.7 19.2 38.9 21.2 39.9 61.1 72 0.04 23.3 19.2 42.5 26.4 31.1 57.5 72 0.05 26.1 20.3 46.4 27.9 25.7 53.6 72 0.10 26.7 19.4 46.1 29.6 24.3 53.9 72 0.50 29.0 26.0 55.0 23.2 21.8 45.0

Referring to Table 3, it can be seen that as the concentration of linoleic acid increases, the production of I and III increases and the production of II and IV tends to decrease. From this, it could be confirmed that the production distribution of the reduction product was changed according to the concentration change of linoleic acid used as a substrate. Therefore, it can be seen that the production distribution of hydroperoxylinoleic acid, an intermediate product, before the reduction step of the reduction product was controlled by the concentration of linoleic acid, the substrate.

Example 7

Lipoxygenase  Control of the amount of enzyme

A reduction product was obtained in the same manner as in Example 4 except that the amount of the lipoxygenase enzyme used was 1.0 μg or 72 μg, and the mass was analyzed after fractionation.

The distribution rate of each reduction resultant according to the amount of the lipoxygenase enzyme is shown in Table 4 below.

LOX1 (μg) LA (mM) Ⅰ 13- (9Z, 11E) -HODE (%) III 9- (10E, 12Z) -HODE (%) Ⅰ + Ⅲ (%) II 13- (9E, 11E) -HODE (%) IV 9- (10E, 12E) -HODE (%) Ⅱ + IV (%) One 0.50 29.3 28.7 58.0 21.0 21.0 42.0 72 0.50 29.0 26.0 55.0 23.2 21.8 45.0

Referring to Table 4, it can be seen that when the amount of the lipoxygenase enzyme is increased, the distribution ratios of I and III decrease and the distribution ratios of II and IV increase. From this, it can be seen that the production distribution of the reduction product varies depending on the amount of lipoxygenase enzyme used. Therefore, it can be seen that the production distribution of hydroperoxylinoleic acid, which is an intermediate product before the reduction step of the reduction product, was controlled by the amount of the lipoxygenase enzyme.

The present invention can control the distribution of various hydroperoxylinoleic acid products that can be used as a starting material of various oxylipin biosynthetic pathways present in plants, thus providing a variety of biosynthetic pathways that can cope with a wide range of stresses on plants. Can provide a foundation to choose from.

Figure 1 shows the reaction product of the hydroperoxidation reaction of linoleic acid using a corn-derived lipoxygenase enzyme.

FIG. 2 is a SDS-PAGE analysis photograph of lipoxygenase enzyme isolated from and purified from E. coli BL21 (DE3) pLysS / pRSETB / LOX1 cells in which corn-derived lipoxygenase is expressed in bacteria and sonicated.

FIG. 3 is a time-dependent UV-Vis spectrum of corn-derived lipoxygenase producing hydroperoxylinoleic acid using linoleic acid as a substrate.

4 is a result of analyzing the distribution (A) of the reduction product obtained in Example 4 and the distribution (B) of the reduction product obtained in Example 5 through SP-HPLC.

5A is a mass spectrometry spectrum of trimethylsilyl 13- (9Z, 11E) -hydroxyoctadecadienoic acid prepared by Example 4. FIG.

5B is a mass spectrometry spectrum of trimethylsilyl 9- (10E, 12Z) -hydroxyoctadecadienoic acid prepared by Example 4. FIG.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Method for preparing hydroxylinoleic acid using lipoxygenase from          Zea mays and the hydroxylinoleic acid prepared by the same <130> HP0849 <150> KR10-2007-0041982 <151> 2007-04-30 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 2845 <212> DNA <213> Zea mays <400> 1 atgttcggga acatcggaaa gatccccatc atcggcgacc tgacgggcag caacaagaat 60 gcgcacctca agggcaacgt ggtgctcgtg cgcaagaccg tgctcggctt ggacgtcacc 120 agcatcgccg gctccctcct cgacggcatc ggcgagttcc tcggccgcgg cgtcacctgc 180 cagcttatca gctccaccgt cgtcgaccct aacaacggca accgcgggaa gttgggcgcg 240 gaggcgagcc tggagcagtg gctgctgaac ccgccgccgc ttctgtccag cgagaaccag 300 ttccgcgtca ccttcgactg ggaggtggag aagcagggca tcccgggcgc catcatcgtc 360 aagaacaacc acgcctccga gttcttcctc aagaccatca ccctcaacga cgtccccggc 420 cacggcacca tcgtcttcgt cgccaactca tggatctacc cgcagtccaa gtaccgctac 480 aaccgcgtct tcttctccaa cgacacgtac ctccccagcc agatgccggc ggcgctgaag 540 ccctaccgcg acgacgagct ccggaacctg aggggcgacg accagcaggg cccgtaccag 600 gagcacgacc gcgtctaccg ctacgacgtc tacaacgacc tgggcctgcc tgacagcggg 660 aacccgcgcc ccgtcctcgg cggcaccaag gagctcccct acccgcgccg ctgccgcacc 720 gggcggaagc ccaccaagag cgaccccaac agcgagagca ggctcacgct ggtcgacggc 780 gacgtctacg tgccgcgcga cgagcgcttc ggccacatca agaagtcgga cttctacggc 840 tacgccatca aggcgctggt gaacgccgtc atcccggcaa tccgcaccta cgtcgacctg 900 tcgcccggcg agttcgactc cttcaaggac atcatgaagc tgtacgaggg cgggatccag 960 ctgcccaaaa taccagccct cgaggacctg cggaagcagt tcccactcga gctcgtcaag 1020 gatgtcctcc cggtcggcgg cgactacctc ctcaagctcc ccatgccgca gatcatcaaa 1080 gaggacaaga caggttggat gacagatgag gagtttggac gggagattct cgccggcgtg 1140 aaccccatgc tcgtcaagcg tctcacggag ttccctccga ggagcagtct tgacccgagc 1200 aagtacggcg accacaccag caccatcagg gaggcggacc tcgagaacaa gctcgagggc 1260 ctgacggtgc agcaggcgct gcacggcaac cggctctaca tcctggacca ccacgacaac 1320 ttcatgccgt tcctggtcag ggtgaacagc ctggagggca acttcatcta cgccaccagg 1380 accgtgctgt tcctgcgcgg cgacggcacg ctggtgccgg tggccatcga gctgagcctg 1440 cccgagctcc gggacggcct gaccaccgcc aagagcaccg tgtacacgcc caagtcgacc 1500 accggcgcgg aggcgtgggt gtggcacctg gccaaggcct acgccaacgt gaacgactac 1560 tgctggcacc agctcatcag ccactggctc aacacccacg ccgtgatgga gccgttcgtg 1620 atcgccacca accggcagct cagcgtgacg caccccgtgc acaagctcct cctgccgcac 1680 taccgtgaca ccatgaacat caactccaac gcgcgccaga tgctcgtcaa cgccggcggc 1740 atcttcgaga ccaccgtctt cccgcgccag tacgcgttcg agatgtcctc cgtcatctac 1800 aaggactgga acttcacaga gcaggctctc cctgacgacc taatcaagag aggcatggcg 1860 gtcgcagacc cgtcgagccc gtacaaggta cggctgctgg tggaggacta cccgtacgcg 1920 tcggacgggc tggccatctg gcacgccatc gagcagtggg tgacggagta cctcgccgtc 1980 tactacccca acgacggcgt gctgcgggcg gacgtggagc tgcaggcgtg gtggaaggag 2040 gcgcgcgagg tcgggcacgc cgacctcaag gacgcgccct ggtggcccaa gatgcagacg 2100 gtggccgagc tggtcaaggc ctgcaccacc atcatctgga tcgcgtcggc gctccacgcg 2160 gccgtcaact tcgggcagta cccgtacgcc gggtacctcc cgaaccgccc gtccgtcagc 2220 cggaagccga tgccggcgcc gggcagcgac gagtacgcgg agctggagcg caagccggag 2280 aaggtgttcg tgcgcaccat caccagccag ttccaggccc tcgtcggcat ctcgctgctg 2340 gagatcctgt ccagccactc ctccgacgag gtgtacctcg gccagcgcga caccaaggag 2400 tggacgtcgg acgccaaggc gcaggaggcg ttcaagcggt tcggcgcgcg gctgaccgag 2460 atcgagaaac gcgtcgtcac catgaacgcg gaccctcgcc tcaagaaccg caacggcccg 2520 gccgagttcc cctacacgct gctctacccc aacacctccg acacgaaggg cgacgccgcc 2580 ggcatcaccg ccaagggcat tccaaacagc atctccattt gagttctgtc tgtctgagct 2640 gaggacgtac ggtcgtcgca ctagattgtt tgcgtttccc cgttccgttc cgtgaagtgt 2700 ggttctcact tgcgcggtat tgtgcaaata tgccaagtac tccttacaga agtcgctacg 2760 tgaggactgt tgtaataagg ctctattctg ttcctcaata atgaagttac cttgtgttca 2820 aaaaaaaaaa aaaaaaaaaa aaaaa 2845 <210> 2 <211> 872 <212> PRT <213> Zea mays <400> 2 Met Phe Gly Asn Ile Gly Lys Ile Pro Ile Ile Gly Asp Leu Thr Gly   1 5 10 15 Ser Asn Lys Asn Ala His Leu Lys Gly Asn Val Val Leu Val Arg Lys              20 25 30 Thr Val Leu Gly Leu Asp Val Thr Ser Ile Ala Gly Ser Leu Leu Asp          35 40 45 Gly Ile Gly Glu Phe Leu Gly Arg Gly Val Thr Cys Gln Leu Ile Ser      50 55 60 Ser Thr Val Val Asp Pro Asn Asn Gly Asn Arg Gly Lys Leu Gly Ala  65 70 75 80 Glu Ala Ser Leu Glu Gln Trp Leu Leu Asn Pro Pro Pro Leu Leu Ser                  85 90 95 Ser Glu Asn Gln Phe Arg Val Thr Phe Asp Trp Glu Val Glu Lys Gln             100 105 110 Gly Ile Pro Gly Ala Ile Ile Val Lys Asn Asn His Ala Ser Glu Phe         115 120 125 Phe Leu Lys Thr Ile Thr Leu Asn Asp Val Pro Gly His Gly Thr Ile     130 135 140 Val Phe Val Ala Asn Ser Trp Ile Tyr Pro Gln Ser Lys Tyr Arg Tyr 145 150 155 160 Asn Arg Val Phe Phe Ser Asn Asp Thr Tyr Leu Pro Ser Gln Met Pro                 165 170 175 Ala Ala Leu Lys Pro Tyr Arg Asp Asp Glu Leu Arg Asn Leu Arg Gly             180 185 190 Asp Asp Gln Gln Gly Pro Tyr Gln Glu His Asp Arg Val Tyr Arg Tyr         195 200 205 Asp Val Tyr Asn Asp Leu Gly Leu Pro Asp Ser Gly Asn Pro Arg Pro     210 215 220 Val Leu Gly Gly Thr Lys Glu Leu Pro Tyr Pro Arg Arg Cys Arg Thr 225 230 235 240 Gly Arg Lys Pro Thr Lys Ser Asp Pro Asn Ser Glu Ser Arg Leu Thr                 245 250 255 Leu Val Asp Gly Asp Val Tyr Val Pro Arg Asp Glu Arg Phe Gly His             260 265 270 Ile Lys Lys Ser Asp Phe Tyr Gly Tyr Ala Ile Lys Ala Leu Val Asn         275 280 285 Ala Val Ile Pro Ala Ile Arg Thr Tyr Val Asp Leu Ser Pro Gly Glu     290 295 300 Phe Asp Ser Phe Lys Asp Ile Met Lys Leu Tyr Glu Gly Gly Ile Gln 305 310 315 320 Leu Pro Lys Ile Pro Ala Leu Glu Asp Leu Arg Lys Gln Phe Pro Leu                 325 330 335 Glu Leu Val Asp Val Leu Pro Val Gly Gly Asp Tyr Leu Leu Lys Leu             340 345 350 Pro Met Pro Gln Ile Ile Lys Glu Asp Lys Thr Gly Trp Met Thr Asp         355 360 365 Glu Glu Phe Gly Arg Glu Ile Leu Ala Gly Val Asn Pro Met Leu Val     370 375 380 Lys Arg Leu Thr Glu Phe Pro Pro Arg Ser Ser Leu Asp Pro Ser Lys 385 390 395 400 Tyr Gly Asp His Thr Ser Thr Ile Arg Glu Ala Asp Leu Glu Asn Lys                 405 410 415 Leu Glu Gly Leu Thr Val Gln Gln Ala Leu His Gly Asn Arg Leu Tyr             420 425 430 Ile Leu Asp His His Asp Asn Phe Met Pro Phe Leu Val Arg Val Asn         435 440 445 Ser Leu Glu Gly Asn Phe Ile Tyr Ala Thr Arg Thr Val Leu Phe Leu     450 455 460 Arg Gly Asp Gly Thr Leu Val Pro Val Ala Ile Glu Leu Ser Leu Pro 465 470 475 480 Glu Leu Arg Asp Gly Leu Thr Thr Ala Lys Ser Thr Val Tyr Thr Pro                 485 490 495 Lys Ser Thr Thr Gly Ala Glu Ala Trp Val Trp His Leu Ala Lys Ala             500 505 510 Tyr Ala Asn Val Asn Asp Tyr Cys Trp His Gln Leu Ile Ser His Trp         515 520 525 Leu Asn Thr His Ala Val Met Glu Pro Phe Val Ile Ala Thr Asn Arg     530 535 540 Gln Leu Ser Val Thr His Pro Val His Lys Leu Leu Leu Pro His Tyr 545 550 555 560 Arg Asp Thr Met Asn Ile Asn Ser Asn Ala Arg Gln Met Leu Val Asn                 565 570 575 Ala Gly Gly Ile Phe Glu Thr Thr Val Phe Pro Arg Gln Tyr Ala Phe             580 585 590 Glu Met Ser Ser Val Ile Tyr Lys Asp Trp Asn Phe Thr Glu Gln Ala         595 600 605 Leu Pro Asp Asp Leu Ile Lys Arg Gly Met Ala Val Ala Asp Pro Ser     610 615 620 Ser Pro Tyr Lys Val Arg Leu Leu Val Glu Asp Tyr Pro Tyr Ala Ser 625 630 635 640 Asp Gly Leu Ala Ile Trp His Ala Ile Glu Gln Trp Val Thr Glu Tyr                 645 650 655 Leu Ala Val Tyr Tyr Pro Asn Asp Gly Val Leu Arg Ala Asp Val Glu             660 665 670 Leu Gln Ala Trp Trp Lys Glu Ala Arg Glu Val Gly His Ala Asp Leu         675 680 685 Lys Asp Ala Pro Trp Trp Pro Lys Met Gln Thr Val Ala Glu Leu Val     690 695 700 Lys Ala Cys Thr Thr Ile Ile Trp Ile Ala Ser Ala Leu His Ala Ala 705 710 715 720 Val Asn Phe Gly Gln Tyr Pro Tyr Ala Gly Tyr Leu Pro Asn Arg Pro                 725 730 735 Ser Val Ser Arg Lys Pro Met Pro Ala Pro Gly Ser Asp Glu Tyr Ala             740 745 750 Glu Leu Glu Arg Lys Pro Glu Lys Val Phe Val Arg Thr Ile Thr Ser         755 760 765 Gln Phe Gln Ala Leu Val Gly Ile Ser Leu Leu Glu Ile Leu Ser Ser     770 775 780 His Ser Ser Asp Glu Val Tyr Leu Gly Gln Arg Asp Thr Lys Glu Trp 785 790 795 800 Thr Ser Asp Ala Lys Ala Gln Glu Ala Phe Lys Arg Phe Gly Ala Arg                 805 810 815 Leu Thr Glu Ile Glu Lys Arg Val Val Thr Met Asn Ala Asp Pro Arg             820 825 830 Leu Lys Asn Arg Asn Gly Pro Ala Glu Phe Pro Tyr Thr Leu Leu Tyr         835 840 845 Pro Asn Thr Ser Asp Thr Lys Gly Asp Ala Ala Gly Ile Thr Ala Lys     850 855 860 Gly Ile Pro Asn Ser Ile Ser Ile 865 870 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tgcagctggt catatggtcg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aagattcgaa ttcagctcag 20  

Claims (13)

Performing a bi-specific hydroperoxidation reaction on linoleic acid using a mixed solution comprising linoleic acid and corn derived lipoxygenase enzyme; Reducing the reaction product using a reducing agent triphenylphosphine (TPP) or NaBH ₄ ; And And separating the reduction product, wherein the concentration of linoleic acid is 0.03 mM to 0.5 mM. The method of claim 1, The lipoxygenase enzyme is a method for producing hydroxylinoleic acid, characterized in that the protein of SEQ ID NO: 2 or a protein having a sequence homology of 70% or more with SEQ ID NO: 2. The method of claim 1, The reaction product was 13- (9Z, 11E) -hydroperoxyoctadecadienoic acid, 13- (9E, 11E) -hydroperoxyoctadecadienoic acid, 9- (10E, 12Z) -hydroperoxyoctadecadiene Acid or 9- (10E, 12E) -hydroperoxyoctadecadienoic acid. delete The method of claim 1, The concentration of the lipoxygenase enzyme is 0.4 µg / ml to 28.8 µg / ml. The method of claim 1, The reduction product was 13- (9Z, 11E) -hydroxyoctadecadenoic acid, 13- (9E, 11E) -hydroxyoctadecadenoic acid, 9- (10E, 12Z) -hydroxyoctadecadienoic acid or 9 -(10E, 12E) -hydroxyoctadecadienoic acid, the process for producing hydroxylinoleic acid. The method of claim 6, When the reducing agent is TPP, 11 to 15 molecules of the 13- (9Z, 11E) -hydroxyoctadecadienoic acid relative to the 10 molecules of 9- (10E, 12E) -hydroxyoctadecadienoic acid, the 13- ( A method for producing hydroxylinoleic acid, wherein 8 to 12 molecules of 9E, 11E) -hydroxyoctadecadienoic acid and 9 to 13 molecules of 9- (10E, 12Z) -hydroxyoctadecadienoic acid are produced. The method of claim 6, When the reducing agent is NaBH ₄ , 15 to 20 molecules of the 13- (9Z, 11E) -hydroxyoctadecadienoic acid with respect to the 10-molecule of 9- (10E, 12E) -hydroxyoctadecadienoic acid, the 13- 9 to 13 molecules of (9E, 11E) -hydroxyoctadecadienoic acid and 14 to 18 molecules of 9- (10E, 12Z) -hydroxyoctadecadienoic acid. The method of claim 1, The hydroperoxidation reaction is a method for producing hydroxylinoleic acid, characterized in that carried out for 10 to 20 minutes at pH 6-8 and temperature 20-30 ℃. The method of claim 1, When the reducing agent is TPP, after the completion of the hydroperoxidation reaction, adding a organic solvent eluting the reaction product, characterized in that it further comprises a step of producing a hydroxylinoleic acid. The method of claim 10, The organic solvent is at least one selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol and mixtures thereof. The method of claim 1, Separating the reduction product is a method for producing hydroxylinoleic acid, characterized in that carried out by HPLC. delete

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