KR101329361B1 - Method for the spectrometric assay of lipoxygenase in reversed micellar system - Google Patents

Abstract

The present invention relates to a novel method for measuring lipoxygenase activity, and since the surfactant which interferes with the detection of conjugated diene in absorbance measurement can be effectively removed using ethanol, The activity of lipoxygenase can be measured accurately and without error.

Description

Method for the spectrometric assay of lipoxygenase in reversed micellar system

The present invention relates to a novel method for measuring lipoxygenase activity, and more particularly, by inducing an enzyme reaction in a reverse micelle system and removing the surfactant by using ethanol after the reaction is completed. It relates to a method capable of measuring the activity of an agent.

Lipoxygenase (linoleate: oxygen oxidoreductase, EC 1.13.11.12) announced in 1928 the presence of carotene oxidase, an enzyme that Bohn and Heas destroy carotene in soybeans. It was first known, and in 1932 it was identified as an enzyme that oxidizes the unsaturated fats contained in soybeans. This enzyme was named lipoxygenase, and in the 1940's it was proved that carotene oxidase and lipooxygenase were the same enzymes.

Lipoxygenase is unsaturated with cis , cis- 1,4 pentadiene ( cis , cis -1,4 pentadiene, -CH = CH-CH ₂ -CH = CH-) structures in the presence of singlet oxygen. As an oxidase having a substrate specificity that acts only on fatty acids, it is present in various plants, and particularly, lipoxygenase, which is present in soybeans in legumes, is known to have high activity.

On the other hand, lipoxygenase oxidizes essential fatty acids in foods to produce odor and odor, and free radicals produced by this enzyme destroy vitamins and proteins. In particular, it causes serious problems in unheated frozen foods such as soybeans and corn, which means that the activity of lipoxygenase is maintained even at low temperatures.

Therefore, there is a need to study a method for inhibiting or destroying the activity of lipoxygenase, which is a factor of reducing the quality of food. This must be preceded.

The activity analysis of lipoxygenase can be broadly classified into two types. One is the spectrophotometric method in which the conjugated diene, a product of the lipoxygenase reaction, is quantified using 234 nm UV rays. ) and the other is re-epoxy agent and O ₂ to the O ₂ is consumed in the kinase reaction quantified using an oxygen electrode (oxygen electrode) - the consumption method (O ₂ -consuming method).

The former method induces a reaction in the emulsification system and quantifies the conjugated diene as it is. Therefore, when the UV-absorbable water-soluble substance is present in the sample, it cannot be directly applied to coenzyme solution. have.

In addition, the latter assay may induce various reactions that consume O ₂ in addition to the mechanism of lipoxygenase, and thus has a disadvantage in that it is limited to the activity analysis of purely purified and purified enzymes.

Rapid screening of lipoxygenase activity of various agricultural products requires an assay that can directly measure the activity of enzymes at the level of crude enzyme-derived liquids without going through long time and costly separation and purification processes. Therefore, a spectroscopic method of quantitatively analyzing the conjugated linoleic acid, which is a product of the lipoxygenase reaction, by using UV rays is preferable, as described above. There is a lot of difficulty in measuring it directly.

Spectroscopy has used a two-phase system consisting of water and lipids and an emulsion system consisting of water, fats and surfactants, but pointed out a small interfacial problem and layer separation between different solvents. Has been. Therefore, a lot of efforts have been made to solve this problem using a reverse micelle system.

Reversed micellar system is a system in which water-soluble particles of several nm size are stabilized by arranging the hydrophobic portion of the surfactant to the outside and the hydrophilic portion to the inside in a nonpolar solvent. When an aqueous solution containing a substance and a nonpolar solvent containing a surfactant suitable for forming a reverse micelle are mixed, the hydrophilic substances are easily penetrated into the aqueous solution of the reverse micelle and are present in a state where no denaturation occurs. (FIG. 1).

The reverse micelle system can induce a rapid reaction compared to the emulsion system by forming a nano-sized droplet, thereby increasing the reaction surface area, and after the completion of the lipoxygenase reaction, the conjugated diene (conjugated diene) Since there is no water-absorbing material absorbing 234 nm in the solvent layer in which) remains, the lipoxygenase activity can be analyzed at the crude enzyme level.

However, there is a problem that needs to be solved in lipoxygenase activity assay using reverse micelles. Sodium dioctyl sulfosuccinate, aerosol-OT, a representative surfactant added to form reverse micelles , AOT) absorbs strongly at 234 nm, the same absorbance as the reaction product conjugated linolenic acid, causing errors in absorbance measurement.

In order to overcome the above problems, the present invention develops a method for removing the surfactant added for reverse micelle formation after the end of the enzymatic reaction, thereby enabling a new method for accurately measuring the activity of lipoxygenase from the preparation. To provide.

The present invention comprises the steps of inducing reverse micelle formation and enzymatic reaction by mixing a sample containing a substrate alpha-linoleic acid, a surfactant and a lipoxygenase in a non-polar solvent ( a); After the enzymatic reaction of step (a), adding and mixing 30-90% (v / v) ethanol to the reaction solution, followed by centrifugation; And after centrifugation of step (b), taking a supernatant to measure the absorbance (c); It provides a method for measuring the activity of lipoxygenase comprising a.

Hereinafter, the lipoxygenase activity measuring method of the present invention will be described in detail by each step.

Step (a): Reverse micelle ( reverse micelle ) Formation reaction and enzymatic reaction induction step

In this step, a sample containing alpha-linoleic acid, a surfactant, and lipoxygenase as a substrate is mixed with a nonpolar solvent to induce reverse micelle formation and enzymatic reaction. .

Typically, a sample containing polar (water-soluble) lipoxygenase is added to a nonpolar solvent with a surfactant and mixed, followed by vigorous stirring to form reverse micelles. By adding alpha-linoleic acid as a substrate to the formed reverse micelles, the lipoxygenase-catalytic reaction is initiated.

Therefore, preferably, a sample containing a lipoxygenase and a surfactant are first added to a nonpolar solvent, and then mixed to form reverse micelles, and later a substrate, alpha-linoleic acid, is added. It is preferable to induce the enzymatic reaction by addition.

The sample containing lipoxygenase used in this step may be derived from a plant containing lipoxygenase, which is a kind of crude enzyme.

As the nonpolar solvent used in this step, various nonpolar solvents including cyclohexane, n -heptane, n -hexane, and hexadecane may be used. Isooctane may be used. It is preferable to use. When isooctane was used as a nonpolar solvent, the activity of lipoxygenase was relatively higher than that of other nonpolar solvents. This means that the detection sensitivity of the lipoxygenase present in the sample (coenzyme solution) is high and the activity measurement can be accurate.

Meanwhile, the surfactant used in this step may be, for example, sodium dioctyl sulfosuccinate (AOT). Sodium dioctyl sulfosuccinate is a compound better known by the commercial name 'aerosol-OT (AOT)' and is the most representative surfactant used for reverse micelle formation.

On the other hand, in this step, mixing can be carried out, for example, using a vortex mixer. The reverse micelle is usually formed by mixing for about 1 minute. The formed reversed micelles have a diameter of about 1 to 2 nm and a reversed micelle system is formed, so that the mixture becomes transparent.

Step (b): add 30-90% (v / v) ethanol to the reaction mixture, mix and centrifuge

In this step, after the enzymatic reaction of step (a), 30 to 90% (v / v) ethanol is further added to the reaction mixture, mixed, and centrifuged.

The surfactant used in step (a) can be removed by this step. In the present invention, the use of a surfactant is essential for the formation of reverse micelles. If the surfactant has a similar absorption wavelength with a conjugated diene, which is a product of an enzymatic reaction, it may cause interference in absorbance measurement and interfere with accurate activity measurement. Can be.

Therefore, the removal of the surfactant is essential for accurate activity measurement, it was confirmed that the surfactant is effectively removed when using 30 ~ 90% (v / v) ethanol as in the present invention. At this time, it was confirmed that the amount of conjugated diene (conjugated diene) remaining in the isooctane layer may vary depending on the ethanol concentration.

On the other hand, in this step (b), the mixing may be performed using a vortex mixer, for example.

On the other hand, the step (b) may preferably further comprise the step of adding the non-polar solvent of step (a) to the reaction solution before the addition of ethanol and mixing. This is a kind of dilution step, in order to facilitate separation of the layer during centrifugation in the following step and to facilitate the measurement of the absorbance.

Step (c): after centrifugation, The supernatant  Collecting and measuring absorbance

This step is a step of measuring the absorbance by taking the supernatant after centrifugation of step (b). At this time, the absorbance can be measured at 234 nm. Centrifugation separates the non-polar solvent layer formed on the upper layer, and collects it to measure absorbance.

Absorbance can be measured using a conventional absorbance meter. When the amount of conjugated linoleic acid that is a reaction product is measured by absorbance, the activity of the enzyme (lipoxygenase) is determined through a standard curve between the absorbance of the conjugated linoleic acid and the enzyme titer. Can be measured.

Since the present invention can effectively remove the surfactant that interferes with the detection of conjugated linoleic acid in absorbance measurement, the effect of accurately measuring the activity of lipoxygenase from the crude solution is exerted.

1 is a schematic diagram of reverse micelles produced in the present invention.
FIG. 2 is a graph showing that AOT, a surfactant, strongly absorbs at a wavelength of 234 nm.
3 is a graph showing the residual amount of conjugated diene (conjugated diene) which is a reaction product present in the isooctane layer and the ethanol layer according to the change in the concentration of ethanol. ●: isooctane layer, ○: ethanol layer.
Figure 4 is a graph showing the change in absorbance of AOT with or without 30% ethanol treatment. ●: control group, ○: 30% ethanol treated group.
5 is a graph showing the effect of R-value ([H ₂ O] / [AOT]) on the activity of lipoxygenase in the reverse micelle system.
Figure 6 is prepared from the soybean-derived lipoxygenase standard reagent (type 1-B) standard enzyme solution with the enzyme concentration set to 0.18 ~ 0.72 μg / mL, the initial reaction rate (activity) according to the lipoxygenase concentration Is the result of measurement. ●: 0.18 μg / mL, ○: 0.36 μg / mL, ▼: 0.54 μg / mL, Δ: 0.72 μg / mL.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the scope of the present invention is not limited to the following Examples and Experimental Examples, but includes modifications of equivalent technical ideas.

Example  1: Surfactant by Ethanol Addition AOT Removal of

In order to develop a lipoxygenase activity assay using a reverse micelle system prepared using AOT as a surfactant, a process of removing AOT, which is a surfactant, from an assay sample after completion of an enzyme reaction is required. This is because AOT, a surfactant, absorbs strongly at 234 nm, which has the same absorbance as conjugated diene, a reaction product (FIG. 2).

Conditions for the solvent to be added to the analytical sample for removing the surfactant, first, the separation of the analyte and the sample, and then have to have the characteristics that do not extract the conjugated diene, a lipoxygenase reaction product from the analyte. Based on the results of preliminary experiments for the selection of a solvent that meets these conditions, it was confirmed whether the conjugated diene was extracted by the addition of various concentrations of ethanol (FIG. 3).

First, the ethanol stock solution (99.5%) was excluded from the experimental group because it was not separated from the isooctane layer. Next, at a concentration of less than 30%, as shown in FIG. 3, the control group (blank test, absorbance 0.78) showed the same level of absorbance within the error range, and it was determined that the conjugated diene was not extracted from the analytical sample. At concentrations below 20%, however, as the opacity of the analyte increased, the error range of the quantitative analysis increased.

Therefore, in the following, 30% ethanol addition was set as experimental conditions, and 30% ethanol addition was performed to verify the ability of removing AOT, which is a surfactant, from the analyte. The experimental group was set up by increasing the AOT concentration of the analytical sample to 2.5 mM intervals up to 15 mM, and the absorbance change according to the presence or absence of 30% ethanol treatment was observed in FIG. 4.

As a result, the control group (●) showed a significant increase in absorbance with increasing AOT concentration, whereas 30% ethanol treatment (○) effectively removed AOT from the isooctane layer at all tested AOT concentrations. (FIG. 4).

Example  2: Reverse micelle system  Used Lipoxygenase  Establishment of reaction condition of activity assay

The purpose of this study was to determine the effect of the type of nonpolar solvent forming the reverse micelle system on the activity of lipoxygenase. Based on the preliminary test results, relative activities were compared for five organic solvents selected using soy-derived lipoxygenase standard reagent (type 1-B).

Experimental results showed that the specific activity was in the order of isooctane>cyclohexane> n -heptane> n -hexane> hexadecane. Compared to the highest inertness of isooctane, all four other organic solvents showed a rapid decrease in relative activity to 40% or less, and isooctane was selected as an optimal organic solvent (Table 1).

Organic solvent Relative activity (%) ± SD ¹⁾ Isooctane 100.00 Cyclohexane 38.61 ± 1.94 n - heptane (n -heptane) 19.15 ± 2.48 n - hexane (n -hexane) 18.80 ± 5.50 Hexadecane 16.75 ± 3.25 ¹⁾ standard deviations

On the other hand, the R-value ([H ₂ O] / [AOT]) is a factor that greatly affects the formation of reverse micelles, the following experiment was to find the optimal R-value for lipoxygenase activity analysis.

Under the condition that the concentration of AOT was fixed at 100 mM, the R-value was set at 5 intervals up to 30 by adjusting the amount of water added to form a reverse micelle system, and then the enzyme inactivity was measured (FIG. 5).

As the R-value increased from 5 to 10, the rapidly increased specific activity decreased gradually as the R-value increased to 20 or more. The R-value representing the specific activity of 0.315 μmol / min.mg protein = 10 The reaction condition of was found to be optimal.

Example  3: Reverse micelle system  Used Lipoxygenase  Validation of Activity Assay

In this example, it was intended to verify the lipoxygenase activity assay developed in the present invention. After preparing the standard enzyme solution from the soybean-derived lipoxygenase standard reagent (type 1-B), the enzyme concentration was set to 0.18-0.72 μg / mL, and the initial reaction rate (activity) of the enzyme was determined according to the lipoxygenase concentration. Measured.

The experiment was performed by the method described below.

① 100 mM AOT was dissolved in isooctane to prepare a stock solution, and 5.0 mL of the stock solution was placed in a 10 mL screw-cap vial.

② 90 μL of phosphate buffer (pH 7.0) containing soybean-derived lipoxygenase type 1-B was added to the vial such that R value = 10.

③ vigorously mixed for 60 seconds to form a transparent reverse micelle system, and the solution was stirred at 800 rpm at 25 ° C. for the following enzymatic reaction.

(4) Alpha-linoleic acid (25 μmol) was added to initiate the enzyme reaction, and the enzyme reaction was maintained while stirring was continued. 200 μL of the reaction solution was collected at regular intervals during the enzyme reaction.

⑤ The collected samples were placed in a vial containing 1.8 mL of isooctane and mixed for 30 seconds to dilute.

⑥ 2.0 mL of 30% ethanol was added, mixed vigorously for 60 seconds, and centrifuged for 10 minutes at a rate of 5,000 × g for layer separation.

⑦ The supernatant (isooctane layer) was collected and the absorbance was measured at 234 nm.

As a result, initial reaction rates of the samples having lipoxygenase concentrations of 0.18, 0.36, 0.54 and 0.72 μg / mL were 0.046, 0.114, 0.197 and 0.268 μmol / min, respectively (FIG. 6).

Except for 0.18 μg / mL, which showed a relatively low coefficient of determination of 0.968, the initial reaction rate was significantly correlated with enzyme concentration.

These results indicate that the lipoxygenase activity assay using the reverse micelle system established from the present invention can measure lipoxygenase activity at the coenzyme level quickly and accurately. This means that it can be effectively applied to the screening of lipoxygenase.

Claims (9)

Mixing a sample containing the substrate alpha -linoleic acid, a surfactant and a lipoxygenase in a nonpolar solvent to induce reverse micelle formation and enzymatic reaction;
After the enzymatic reaction of step (a), further adding 30% (v / v) ethanol to the reaction mixture, mixing and centrifuging (b); And
After centrifugation of step (b), taking a supernatant to measure the absorbance (c); Method for measuring the activity of lipoxygenase comprising a.
The method of claim 1,
The step (a)
Samples and surfactants containing lipoxygenase are first added to a nonpolar solvent, followed by mixing to form reverse micelles, followed by the addition of the substrate, alpha-linoleic acid, to undergo enzymatic reaction. Induction method for measuring the activity of lipoxygenase.
The method of claim 1,
The non-
A method for measuring activity of lipoxygenase, characterized in that it is isooctane.
The method of claim 1,
The surfactant is,
A method for measuring the activity of lipoxygenase, characterized in that it is sodium dioctyl sulfosuccinate (AOT).
The method of claim 1,
Mixing of the step (a),
Method for measuring the activity of lipoxygenase, characterized in that carried out through a vortex mixer.
The method of claim 1,
Mixing of the step (b),
Method for measuring the activity of lipoxygenase, characterized in that carried out through a vortex mixer.
The method of claim 1,
Samples containing lipoxygenase,
A method for measuring activity of lipoxygenase, which is derived from a plant containing lipoxygenase.
The method of claim 1,
The absorbance of step (c) is
Method for measuring the activity of lipoxygenase, characterized in that measured at 234 nm.
The method of claim 1,
The step (b)
The method of measuring the activity of lipoxygenase further comprising the step of adding and mixing the nonpolar solvent of step (a) to the reaction solution before the addition of ethanol.

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