JP2003274943A - Glutathione peroxidase enzyme and method for assaying activity of glutathione peroxidase enzyme - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new glutathione peroxidase having excellent stability with slight lowering of an activity during solution preservation and to provide a method for assaying the activity of the glutathione peroxidase with which actions on a lipid peroxide can directly be evaluated with a high assaying sensitivity as the method for assaying the activity of the glutathione peroxidase enzyme associated with removal of the lipid peroxide in vivo. <P>SOLUTION: The glutathione peroxidase is obtained from a plant, a bacterium, algae, a mold or a mushroom. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel glutathione peroxidase enzyme. The present invention also relates to a method for directly and highly sensitively measuring glutathione peroxidase enzyme activity.

[0002]

2. Description of the Related Art Glutathione peroxidase enzyme is
It is one of the enzymes involved in the removal of lipid peroxide in the living body. Lipid peroxide is generated in the tissues of living organisms due to various causes. The increase in active enzymes produced by the decomposition of lipid peroxide is involved in so-called lifestyle-related diseases such as arteriosclerosis and diabetes, and diseases related to aging such as cataract. There are various enzyme systems that decompose and remove active enzymes in the living body, and the glutathione peroxidase enzyme is one of them and is an enzyme that catalyzes the reaction of the formula 1 shown below. The glutathione peroxidase enzyme has a function of reducing and removing lipid peroxide which is a source of active oxygen production, and is a peroxidase that requires reduced glutathione as a hydrogen donor in the reaction (Watabe Rei・ Akira Hiratsuka: Protein Nucleic Acid Enzyme (Temporary Sensitization) -Reactive Oxygen ・ Molecular mechanism of generation, elimination and action in living organisms,
2949-2956 (1988)).

A method for measuring the activity of glutathione peroxidase enzyme has conventionally been carried out by a method called a coupled enzyme method utilizing a secondary reaction by combining the reactions shown in the following formulas 1 and 2. (Litt
Le et al. Biol. Chem. 245, 36
32 (1970)). That is, a water-soluble substance such as t-butyl hydroperoxide or hydrogen peroxide is used as the peroxide on the left side of Formula 1, and the hydroxy product (water in the case of hydrogen peroxide) as a reaction product is quantified. As shown in Formula 2, it is difficult to make nicotinamide adenine dinucleotidolinic acid (NADP) by using glutathione reductase coexisting in the reaction solution.
H) is a method of measuring the decrease amount, and the measurement of the decrease amount of NADPH is performed by measuring the decrease in absorbance at a fixed wavelength.

[0004]

[Chemical 1]

It has been known that glutathione peroxidase enzyme is distributed in animal blood, various organs, and microorganisms of the genus Hannucella (Takeshi Watanabe, Akira Hiratsuka: Protein Nucleic Acid Enzyme (Temporary Sensitization) -Active Oxygen). Molecular mechanism of generation, elimination and action in organisms, 33, 2949 to 2956 (1
988), and JP-A-4-94682), partially purified products derived from bovine platelets and human platelets are industrially produced. However, these are problematic in that the obtained amount is small and that the protein is easily denatured and inactivated in a solution, and thus a novel glutathione peroxidase enzyme having high activity stability has been demanded.

[0006] Further, in the above-mentioned method for measuring the activity of glutathione peroxidase enzyme, glutathione peroxidase enzyme is indirectly measured, since the decrease amount of NADPH, which decreases with the two-step reaction, is measured. Can be said. Therefore, as a result, N
When it is presumed that a system for regenerating ADPH exists, there is a problem that the activity cannot be accurately measured. In addition, when it is attempted to measure the action strength against lipid peroxide in consideration of the in vivo function of glutathione peroxidase enzyme, t-butyl hydroperoxide, which is a synthetic substance that cannot exist in the body, is used as a substrate. Is not suitable for use as. Furthermore, theoretically, measurement can be performed if the enzyme is contained in 1 milliliter (mL) of the solution in an amount of 0.1 unit or more, but in practice, if the measurement sample is turbid, the background will rise, and
If it is more than unit, it is difficult to measure, and the glutathione peroxidase enzyme to be measured originally has considerably high activity, or it is impossible to make accurate measurement unless the sample solution is partially purified and used. there were.

[0007]

The present invention has been made in view of such conventional problems, and provides a novel glutathione peroxidase enzyme excellent in stability with little decrease in activity during solution storage. The purpose is to do. Further, the present invention, as a method for measuring the activity of glutathione peroxidase enzyme involved in the removal of lipid peroxide in vivo, the activity of glutathione peroxidase enzyme, which can directly evaluate the action on lipid peroxide, and has high measurement sensitivity. The purpose is to provide a measuring method.

[0008]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that glutathione peroxidase enzyme can be obtained from a raw material whose existence has not been known in the past. It has been found that the glutathione peroxidase enzyme is a novel enzyme and exhibits excellent stability with little decrease in activity during solution storage. Furthermore, the present inventors have found that it is possible to directly quantify the hydroxy form of the reaction product by using lipid peroxide as the substrate peroxide. According to this method, not only is it possible to appropriately evaluate the enzyme action of glutathione peroxidase on lipid peroxides in vivo, but it is also possible to measure glutathione peroxidase enzyme activity with extremely high sensitivity in order to directly quantify reactive organisms. It becomes possible to do.

That is, the present invention relates to plants, bacteria, algae,
Provided is a glutathione peroxidase enzyme obtained from mold or mushroom.

The present invention also provides a polar organic solvent treatment, salting-out, ultrafiltration treatment, adsorption chromatography of an extract containing glutathione peroxidase enzyme obtained from plants, bacteria, algae, fungi, or mushrooms. Provided is a glutathione peroxidase enzyme obtained by an ion exchange chromatography or a gel filtration operation, or by purifying it by combining two or more thereof.

The glutathione peroxidase enzyme of the present invention is characterized by having a molecular weight of 30,000 to 80,000.

The present invention also provides a reaction solution obtained by reacting a glutathione peroxidase enzyme with a solution containing lipid peroxide and reduced glutathione as main components.

The lipid peroxide is preferably linoleic acid hydroperoxide or linolenic acid hydroperoxide.

In the reaction solution of the present invention, plants,
It is preferable to use glutathione peroxidase enzyme obtained from bacteria, algae, fungi, or mushrooms, and an extract containing glutathione peroxidase enzyme extracted from the above raw material may be used in the form of a crude enzyme solution without purification. Alternatively, it may be used in a purified form by treatment with a polar organic solvent, salting out, ultrafiltration treatment, adsorption chromatography, ion exchange chromatography or gel filtration operation, or a combination of two or more thereof.

Furthermore, in the reaction solution of the present invention, the lipid peroxide concentration is 0.001 to 10 mmol / liter, the reduced glutathione concentration is 0.005 to 50 mmol / liter, and the lipid peroxide is It is preferable that the concentration ratio of the product to reduced glutathione is 1: 1 to 1:20.

Further, the present invention comprises a step of removing a water-soluble substance from the reaction solution using an organic solvent and quantifying a product hydroxy compound by using a high performance liquid chromatograph. Provide a measurement method.

The eluate of the high performance liquid chromatograph preferably contains hexane, 2-propanol and acetic acid as main components, and the volume ratio thereof is 95 to 99: 5 to 1: 0.
It is preferably from 01 to 1.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention provides a novel glutathione peroxidase enzyme obtained from plants, bacteria, algae, fungi and mushrooms (hereinafter also referred to as "glutathione peroxidase enzyme of the present invention"). . Examples of the plant include leeks and the like. More specifically, the leeks include long leeks and allium varieties. Examples of algae include wakame and kelp, and examples of mushrooms include shiitake, shimeji mushroom, and enoki mushroom. It was first discovered by the present inventors that glutathione peroxidase can be obtained from these raw materials, and as will be described later, the glutathione peroxidase enzyme of the present invention is a novel one and has little activity reduction during solution storage. It is extremely stable. Incidentally, the glutathione peroxidase enzyme of the present invention, unless otherwise specified, plants, bacteria,
As it is extracted from algae, mold, or mushroom, it may be in the form of a crude enzyme solution without being purified, or it may be in the form of being purified by a known means described below after extraction, and glutathione of the present invention. The form is not limited as long as it contains a peroxidase enzyme and has glutathione peroxidase activity.

The extraction conditions for the glutathione peroxidase enzyme of the present invention will be described. First, a plant, bacterium, algae, mold or mushroom (hereinafter, also referred to as “raw material of the present invention”), which is an extraction raw material in the present invention, is homogenized while cooling in water or a buffer solution to obtain a ground solution of the raw material. . The pH of the water or buffer used, the cooling temperature and the homogenization time can be set appropriately.

For example, it is usually about 1 to 10 parts by mass, preferably about 3 parts per 1 part by mass (wet mass) of the raw material of the present invention.
A homogenizer made of stainless steel (for example, Ultra-d manufactured by Yamato Scientific Co., Ltd.) is added by adding ~ 8 parts by mass of water or a buffer solution.
isperser LK-21, cutter S25N-1
8G) while cooling to 0-5 ° C for 0.5-1 minute,
Homogenate. When the temperature in the homogenate is below 0 ° C, the solution freezes, and when it exceeds 5 ° C, the mixture during grinding is heated by mechanical heat in the homogenate, and the activity of glutathione peroxidase enzyme contained in the mixture is lost. There is a case to be seen. If the homogenation time is less than 0.5 minutes, the homogenization is not sufficient, and if it exceeds 1 minute, the glutathione peroxidase enzyme activity may be inactivated by heating. The pH of the added water or buffer is 5-9.
Is preferable, and more preferably pH is 7.5 to 8.5. If the pH is outside the range of 5 to 9, glutathione peroxidase enzyme activity may be inactivated, which is not preferable.

The plants, bacteria, algae, molds and mushrooms as the raw material for extracting the glutathione peroxidase enzyme of the present invention may be raw ones, but the dried products obtained by removing water by freeze-drying and the like can also be used. The enzyme can be extracted.

Then, the pulverized mixture thus obtained is centrifuged to remove fat, and the aqueous layer is filtered with a filter aid such as diatomaceous earth or cellulose to obtain the crude enzyme. Get the liquid. This crude enzyme solution is clear.

The resulting crude enzyme solution is used, if necessary, by a known means such as treatment with a polar organic solvent, salting out, ultrafiltration treatment, adsorption chromatography, gel filtration operation, ion exchange chromatography, or the like. Two or more kinds can be combined and separated and purified as a single component. In the examples described below, the case of using a polar organic solvent treatment, gel filtration operation, and ion exchange chromatography is illustrated, but salting out, ultrafiltration treatment, and adsorption chromatography are described, for example, in “Biochemistry Laboratory Course”. 1-Chemistry of Protein I ”, Nobuo Ui, Nobuo Tamiya, Kozo Narita, 59
Page, page 68, page 133 (1976), Tokyo Kagaku Dojin, etc.

The previously known glutathione peroxidase enzyme is derived from bovine platelets and has a molecular weight of 85.0.
Around 00 (by gel filtration method. VF Schneider et al., Ho
ppe-Seyler's Physoil. Chem. 348, 540 (see 1967), bovine erythrocyte-derived around 84,000 (SD
By S-polyacrylic gel electrophoresis. L. Flohe et al.,
Hoppe-Seyler's Physoil. Chem. 352, 151 (1971)
, And the raw material of the present invention, which is greater than 80,000,
That is, the molecular weight of the glutathione peroxidase of the present invention obtained from plants, bacteria, algae, molds or mushrooms was measured by SDS-polyacrylamide gel electrophoresis and found to be 30,000 to 80,000.

Next, a glutathione peroxidase enzyme activity measuring method (hereinafter, simply referred to as "activity measuring method of the present invention").
Also say. ) Will be described. The activity measuring method of the present invention is not limited to the activity measurement of the novel glutathione peroxidase enzyme of the present invention, and can be suitably used for the activity measurement of known glutathione peroxidase enzyme such as commercially available bovine platelets. You can

In the activity measuring method of the present invention, a lipid peroxide is used as a substrate, and the production amount of a hydroxy form converted from the lipid peroxide by the catalytic action of glutathione peroxidase is directly measured by liquid chromatography (HPLC). The enzymatic activity of glutathione peroxidase is determined by quantitative measurement. When a water-soluble substance such as t-butyl hydroperoxide or hydrogen peroxide is used as a substrate, it is difficult to quantify the hydroxy product which is a reaction product, and thus it has hitherto been a coupled enzyme utilizing a secondary reaction. The glutathione peroxidase activity could be measured only by the method, but according to the present invention, the hydroxy form produced by the conversion of the lipid peroxide used as the substrate is easy to quantify, and therefore the glutathione peroxidase enzyme activity is It becomes possible to measure glutathione peroxidase enzyme activity directly and with high sensitivity by utilizing the correlation between the amount of hydroxy form and the amount of hydroxy form produced. Furthermore, by using lipid peroxide as a substrate, in vivo glutathione peroxidase enzyme can be measured. It has become possible to more appropriately evaluate the action in.

That is, in the activity measuring method of the present invention, a lipid peroxide is used as the peroxide (R-OOH) shown on the left side of the above formula 1, and the lipid peroxide and reduced glutathione (GSH) are used. The glutathione peroxidase enzyme activity in the sample is determined by measuring the production amount of the hydroxy form obtained by reacting the mixture with glutathione peroxidase enzyme. The lipid peroxide as a substrate in the present invention is not particularly limited, and examples thereof include linoleic acid hydroperoxide and linolenic acid hydroperoxide. The form of the glutathione peroxidase enzyme used as the sample is not particularly limited as long as it contains glutathione peroxidase enzyme activity, and an aqueous solution containing the isolated glutathione peroxidase enzyme is, of course, extracted from the plant. Even a solution containing a component other than the glutathione peroxidase enzyme, such as a liquid, may contain glutathione peroxidase enzyme activity.

The activity measuring method of the present invention will be specifically described below. First, the step of converting a peroxide into a hydroxy form by the catalytic action of glutathione peroxidase enzyme will be described.

Lipid peroxide is converted into a hydroxy form by reacting a mixture of lipid peroxide and reduced glutathione with a glutathione peroxidase enzyme. Usually, each component is converted into an appropriate buffer solution such as phosphate buffer. It is used by dissolving it in liquid, Good's buffer, Tris buffer, etc. The pH of the buffer solution is preferably 5 to 9 and more preferably pH 7.5 to 8.5 from the viewpoint of not deactivating the glutathione peroxidase enzyme activity.

The liquid obtained by reacting a mixture of lipid peroxide and reduced glutathione with glutathione peroxidase enzyme is referred to as "reaction liquid" in the present specification. The lipid peroxide concentration at the time of preparing the reaction solution is preferably 0.00
It is 1 to 10 mmol / liter. If the lipid peroxide concentration is less than 0.001 mmol / liter, it may be difficult to detect the product hydroxy compound because it is hidden by background noise. On the other hand, if it exceeds 10 mmol / liter, the reaction does not proceed quantitatively, and the relationship between the glutathione peroxidase enzyme activity in the sample and its measured value tends to show no linearity. Therefore, the concentration of lipid peroxide is more preferably 0.02 to 0.05 mmol / liter. The concentration of reduced glutathione in the reaction solution at the time of preparing the reaction solution is preferably 0.005 to
50 mmol / l. If the concentration of reduced glutathione is less than 0.05 mmol / liter, the concentration may be too low to function sufficiently as a coenzyme.
On the other hand, when it exceeds 50 mmol / liter, the spontaneous reduction reaction of lipid peroxide by reduced glutathione cannot be ignored. Therefore, the concentration of reduced glutathione is more preferably 0.1 to 0.5 mmol / liter. Furthermore, the concentration ratio of lipid peroxide and reduced glutathione at the time of preparing the reaction solution is preferably 1: 1 to 1: 2.
It is 0. If the concentration ratio of reduced glutathione is lower than 1: 1, it may not function sufficiently as a coenzyme, while if reduced glutathione is present at a concentration ratio higher than 1:20, spontaneous lipid peroxidation occurs. The reduction reaction of substances cannot be ignored. Therefore, each concentration ratio is 1: 5
It is more preferably 1:15.

In this step, the reaction liquid is preferably 5 to 60.
By heating at 5 ° C, more preferably 28 to 30 ° C for 5 to 10 minutes, a hydroxy compound as a product is obtained. At this time, if the temperature is less than 5 ° C or the reaction time is less than 5 minutes, the progress of the reaction is insufficient, while if the temperature exceeds 60 ° C or the reaction time exceeds 10 minutes, the enzyme is inactivated. It is not preferable because it may occur. After the reaction time has elapsed, the reaction solution is acidified to pH 4.0 to stop the reaction.

Next, the step of measuring the hydroxy product as a product will be described. In this step, the hydroxy form is extracted from the reaction solution using an organic solvent, and the hydroxy form is directly measured using high performance liquid chromatography (HPLC). As the organic solvent for extracting the hydroxy form,
There is no particular limitation as long as it is insoluble in water.
For example, a chloroform-methanol mixed solution, chloroform, hexane, benzene, or the like can be used alone or as a mixture of two or more kinds. From this organic solvent mixture, an organic solvent layer is obtained by centrifugation or a separating funnel. The obtained organic solvent layer is concentrated under reduced pressure, and this is subjected to chromatography as a hexane solution.

The hexane solution of the concentrate of the organic solvent layer contains not only the hydroxy product as a product but also unreacted lipid peroxide, which interferes with the ultraviolet absorption of the product. Therefore, it is necessary to measure separately. As a method for measuring the production amount of the hydroxy form, a high performance liquid chromatograph connected to a normal phase silica column was used, and a hexane: 2-propanol: acetic acid mixture (preferably 95 to 99: 5) was used as a high performance liquid chromatograph eluent. ~ 1: 0.01 ~
1 (v / v), more preferably 97-99: 3-1:
0.05 to 0.2) can be used to quantify the hydroxy form of the reaction product while detecting at 230 to 240 nm, preferably 232 to 235 nm. The volume ratio of hexane: 2-propanol in the eluate is less than 95%,
More than 5%, or more than 99% and less than 1% respectively,
It becomes difficult to separate the lipid peroxide which is the substrate and the hydroxy form which is the product. Further, if the acetic acid concentration ratio is less than 0.01%, it becomes difficult to separate the lipid peroxide and hydroxytite, and if the concentration ratio exceeds 1%, the background noise increases and the sensitivity decreases. Also, the detection wavelength is 2
If it is less than 30 nm or exceeds 240 nm, background noise increases and sensitivity decreases.

Then, the glutathione peroxidase enzyme activity may be determined by a known means from the measured production amount of the hydroxy form. As a method for calculating the glutathione peroxidase enzyme activity from the amount of hydroxy form produced, for example, a calibration curve is prepared in advance using a glutathione peroxidase enzyme standard solution having a known activity value, and the calibration curve in the sample is used using this calibration curve. The glutathione peroxidase activity can be determined, or the hydroxy body production amount can be calculated from the area value shown in the chart of the high performance liquid chromatograph, and the hydroxy body 1
Since 2 moles of reduced glutathione are consumed when producing moles, the amount of enzyme that consumes 1 micromol of reduced glutathione per minute can be calculated as 1 unit.

[0035]

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of Glutathione Peroxidase Enzyme-Containing Extract Liquid An extract liquid containing the glutathione peroxidase enzyme of the present invention was prepared by the following procedure. A long onion leaf is used as a raw material, 0.05M Tris buffer solution (pH 8.0) in an amount 4 times as much as the (wet) mass ratio of the long onion leaf is added, and a homogenizer (manufactured by Yamato Scientific Co., Ltd., Ultra-
disperser, LK-21, cutter S25N-1
8G) for 0.5 minutes and then 10,000 rpm for 10
Centrifugation was carried out for a minute, and the supernatant was added to Toyo No.
By filtering with 2 filter paper and collecting the filtrate, an extract containing the glutathione peroxidase enzyme of the present invention was obtained. All the above operations were performed at 5 ° C. It was examined in Example 2 and below that the filtrate contained the glutathione peroxidase enzyme of the present invention.

Example 2 Activity Measurement of Glutathione Peroxidase Enzyme Obtained in Example 1 A linoleic acid hydroperoxide solution was used as a lipid peroxide serving as a substrate for measuring glutathione peroxidase enzyme activity. 3 reaction liquids having the composition shown in Table 1
After incubating at 0 ° C for 5 minutes, chloroform-methanol =
3 ml of an organic solvent consisting of 2: 1 (v / v) was added and the organic solvent layer was recovered by centrifugation to obtain unreacted linoleic acid hydroperoxide and a reaction product hydroxy compound mixed solution.

Next, this solution was subjected to a vacuum distillation apparatus to remove the organic solvent. The obtained concentrate was dissolved in hexane and used as a column for Inertsil SIL (4.6 ×).
150 mm, GL science Co. LTD) was connected to the column and subjected to HPLC in which the column temperature was set to 35 ° C., and hexane: 2-propanol: acetic acid = 99: 1: 0.1 (v / v / v) (ml / min) as an eluent. Was used to separate and quantify the reaction product hydroxy form while detecting at 235 nm.

[0039]

[Table 1]

The unit of activity is calculated by calculating the amount of hydroxy form produced from the area value shown in the HPLC chart, and 2 mol of reduced glutathione is consumed when 1 mol of hydroxy form is produced. The amount of enzyme that consumes reduced glutathione was set to 1 unit. According to the activity measuring method of the present invention, glutathione peroxidase activity in the leaf part of the long onion was 0.138 units / g (Table 4).

(Example 3) Purification of glutathione peroxidase enzyme Acetone was gradually added to 430 mL of the extract obtained from 100 g of leek leaves by the method of Example 1 so that the volume ratio of acetone to the total volume was 30%. Then, it was allowed to stand at 4 ° C for 1 hour. Then, the precipitate was removed by centrifugation, and acetone was added to the separated liquid in a volume ratio of 6 to the total volume.
The mixture was gradually added to 0%, left standing at 4 ° C. for 1 hour and then centrifuged to obtain a precipitate. The precipitate was adjusted to pH 8.
It was dissolved in 40 mL of 0 Tris buffer. The specific activity of the glutathione peroxidase enzyme contained in this enzyme solution was 0.241 unit per the amount of protein contained.
/ Mg protein.

The above enzyme solution was further added with a diameter of 3.0 cm × 9.
The sample was applied to a 0 cm Sephadex G-100 gel filtration column chromatograph (Pharmacia High-Tech),
A 20 mL fraction of a solution having enzyme activity was obtained. The specific activity of glutathione peroxidase enzyme contained in the obtained fraction was 3.12 units / per unit of the amount of protein contained.
It was mg protein.

The glutathione peroxidase enzyme solution obtained from the above-mentioned column chromatograph was concentrated 10 times with an ultrafiltration membrane (Morcut II: manufactured by Japan Millipore), and 0.01M phosphate buffer solution was prepared. (P
H6.0) 1.0cm x 20cm diameter equilibrated with
CM-Sephadex C-50 cation exchange column chromatograph (manufactured by Pharmacia Biotech), and a gradient of sodium chloride concentration 0 to 0.1M with the same buffer is applied to obtain a solution of 10 mL of a fraction having the enzyme activity. Obtained. The specific activity of the glutathione peroxidase enzyme contained in the obtained fraction was 9.26un per the amount of protein contained.
It was ts / mg protein.

Example 4 Enzymatic Chemical Properties of Glutathione Peroxidase of the Present Invention Extracted and Purified from Long Allium Leaves In Example 3 to show that glutathione peroxidase extracted from long onion leaves is a novel glutathione peroxidase of the present invention. Various properties of the obtained protein as an enzyme were examined.

1) Molecular weight of enzyme: The molecular weight by SDS-polyacrylamide gel electrophoresis is about 46,000 Da.
Met.

2) Coenzyme: The reaction occurred only when reduced glutathione was added, and the reaction did not occur with NAD (P) H, ascorbic acid, mercaptoethanol, ethyl mercaptan and cysteine. Therefore, the presence of glutathione peroxidase was shown in the purified product extracted and purified from the leek leaf. In addition, the same glutathione peroxidase enzyme was detected in the leaves, the central part, and the skin of long onions.

3) Optimum pH and stable pH: pH 8.0
Showed the maximum activity. Further, after being dissolved in a buffer solution having a pH range of 3 to 12 and allowed to stand at 40 ° C. for 10 minutes, 80% activity was retained in the pH range of 5 to 9. Therefore, it was stable in the range of pH 5-9.

4) Optimum temperature: The action was optimum at a temperature of about 30 ° C.

5) Thermal stability: Deactivated when heat-treated at 60 ° C. for 20 minutes, and deactivated within 5 minutes at 75 ° C. or higher.

6) Freezing resistance: It does not inactivate even if it is frozen without adding a stabilizer, and can be stored for 3 months or more.

(Example 5) Measurement of glutathione peroxidase enzyme activity (1) Regarding the method for measuring glutathione peroxidase enzyme activity, the present invention was compared with the conventional method using glutathione peroxidase derived from bovine platelets (Table 3). . The activity of glutathione peroxidase enzyme according to the present invention was measured in the same manner as in Example 2. The activity of glutathione peroxidase enzyme was measured by the conventional method (coupling enzyme method using t-butyl hydroperoxide as a substrate) by measuring the decrease in absorbance at 340 nm of the reaction solution having the composition shown in Table 2. In addition, the conventional method by the coupled enzyme method using hydrogen peroxide and linoleic acid hydroperoxide as substrates was also examined (Table 3). The unit of activity is 1 micromolar NAD per minute.
The amount of enzyme that produces P was 1 unit.

The same sample of glutathione peroxidase enzyme derived from bovine platelets was dissolved in 0.1 M Tris buffer to each concentration shown in Table 3, and the glutathione peroxidase enzyme activity in the solution was measured according to the present invention and the conventional method. Table 3 shows the results measured by the method. In the conventional method and the method of the present invention, the activity value of glutathione peroxidase enzyme, that is, the unit / mL number showed almost the same value. However, it can be seen that the measurement sensitivity is improved about 100 times in the present invention as shown in Table 3 while the conventional method cannot be measured when the unit unit is 0.3 or less.
In addition, the activity measurement method of the present invention was found to have improved sensitivity even compared to the conventional method using linoleic acid hydroperoxide as a substrate. It was brought about by directly quantifying the hydroxy form of the product, and it was shown that the extremely important effect of the present invention is found in that the hydroxy form of the product can be directly measured. .

[0053]

[Table 2]

[0054]

[Table 3]

(Example 6) Measurement of glutathione peroxidase enzyme activity (2) Table 4 shows glutathione peroxidase activity measured by the activity measuring method of the present invention and the conventional method for the raw material of the present invention such as plants. The activity according to the present invention was measured in the same manner as in Example 2, and the conventional method was carried out in the same manner as in Example 5 using t-butylhydroperoxide as a substrate. For the extracts from plants, bacteria, seaweeds, molds and mushrooms shown here, glutathione peroxidase activity was not measured by the conventional method, but activity was recognized by the method of the present invention. When the conventional method is used, the turbidity of the sample extract from these raw materials affects the measurement sensitivity, which is lower than the above-described sensitivity, and the detection sensitivity per 1 g of wet sample mass is 1 uni.
Although it was limited to t or more, it was 0.001 in the method of the present invention.
It was possible to detect up to unit / g.

[0056]

[Table 4]

[0057]

INDUSTRIAL APPLICABILITY According to the present invention, a novel glutathione peroxidase is provided from a raw material which has not been known so far. This novel glutathione peroxidase has a higher activity stability than known bovine platelet-derived and human platelet-derived ones, and the storage and management of the reagents are easy. Since such a raw material may be a dried product, for example, it becomes possible to make an effective resource for the waste part of leeks.

The present invention also provides a method for measuring glutathione peroxidase activity, which enables direct evaluation of glutathione peroxidase action on lipid peroxides. This method makes it possible to increase the measurement sensitivity from the conventional level of about 1 unit / mL to about 0.001 unit / mL.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Junji Terao             3-18-15 Kuramoto-cho, Tokushima City, Tokushima Prefecture University of Tokushima             Faculty of medicine F-term (reference) 4B050 CC01 DD02 DD03 DD13 FF01C                       FF04C FF05C FF09C FF11C                       FF12C LL03                 4B063 QA01 QQ22 QR45 QR48 QR50                       QX01                 4H055 AA01 AA03 AB37 AC60 AD32                       BA50 CA10 CA60

Claims (13)

[Claims] 1. A glutathione peroxidase enzyme obtained from plants, bacteria, algae, molds or mushrooms. 2. A polar organic solvent treatment, salting out, ultrafiltration treatment of an extract containing glutathione peroxidase enzyme obtained from plants, bacteria, algae, molds or mushrooms,
A glutathione peroxidase enzyme obtained by adsorption chromatography, ion exchange chromatography, gel filtration operation, or purification by combining two or more thereof. 3. The glutathione peroxidase enzyme according to claim 1 or 2, having a molecular weight of 30,000 to 80,000. 4. A reaction solution obtained by reacting a glutathione peroxidase enzyme with a solution containing lipid peroxide and reduced glutathione as main components. 5. The reaction solution according to claim 4, wherein the lipid peroxide is linoleic acid hydroperoxide or linolenic acid hydroperoxide. 6. A glutathione peroxidase enzyme,
The reaction liquid according to claim 4 or 5, which is obtained from a plant, a bacterium, an alga, a mold, or a mushroom. 7. A glutathione peroxidase enzyme,
An extract obtained from plants, bacteria, algae, fungi, or mushrooms, which contains glutathione peroxidase enzyme extracted from plants, bacteria, algae, fungi, or mushrooms, is used as a crude enzyme solution without purification. Used in the form of
The reaction liquid according to claim 4 or 5. 8. A glutathione peroxidase enzyme,
An extract obtained from a plant, bacteria, algae, mold, or mushroom, which is extracted from the plant, bacteria, algae, mold, or mushroom, and which contains a glutathione peroxidase enzyme, is treated with a polar organic solvent, and salted out. The reaction solution according to claim 4 or 5, which is used in a purified form by an ultrafiltration treatment, an adsorption chromatography, an ion exchange chromatography or a gel filtration operation, or a combination of two or more thereof. 9. The method according to claim 6, wherein the glutathione peroxidase enzyme has a molecular weight of 30,000 to 80,000.
8. The reaction liquid according to any one of 8 above. 10. The lipid peroxide concentration is 0.001-10.
Mmol / liter, reduced glutathione concentration is 0.0
The concentration ratio of the lipid peroxide and the reduced glutathione is from 1: 1 to 0.5 to 50 mmol / liter.
The reaction liquid according to any one of claims 4 to 9, which is 1:20. 11. A process of removing a water-soluble substance from the reaction liquid according to claim 4 by using an organic solvent, and quantifying a hydroxy product as a product by high performance liquid chromatography. A method for measuring the activity of glutathione peroxidase enzyme, which comprises: 12. An eluate of a high performance liquid chromatograph containing hexane, 2-propanol and acetic acid as main components.
The activity measuring method according to claim 11. 13. The activity measuring method according to claim 12, wherein the volume ratio of hexane, 2-propanol and acetic acid is 95-99: 5-1: 0.01-1.