JPH0556780A - Laccase inhibitor and usage thereof - Google Patents

Abstract

PURPOSE:To use hadacidin and/or N-hydroxyglycine as a herbicide, a browning inhibitor, a cosmetics and an inhibitor for laccase action of bilirubin oxidase CONSTITUTION:Hadacidin and/or N-hydroxyglycine is applied to a herbicide, a food-browning inhibitor and a cosmetics as a melanin pigment production inhibitor. In addition, interference to analysis of bilirubin can be avoided or reduced by using these substances in combination with bilirubin oxidase in the field of clinical examination since the reaction of bilirubin oxidase on the chromogen can be inhibited without inhibiting the reaction on bilirubin in an oxidase-peroxidase-chromogen system.

Description

Detailed Description of the Invention

[0001]

This invention relates to laccase inhibitors and their uses. More particularly, with respect to hadacidin or N-hydroxyglycine as laccase inhibitors,
Further, the present invention relates to the use of the inhibitor as a herbicide, an agent for preventing browning, a cosmetic, or a reagent for clinical examination.

Specifically, for example, it relates to use as a herbicide for wormwood and the like, an agent for preventing browning of vegetables such as potatoes and apples, a skin cosmetic, and a laccase action inhibitor of bilirubin oxidase used in clinical tests.

As a herbicide, an anti-browning agent and a skin cosmetic, the action as a laccase action inhibitor is utilized. Also,
As a use in the field of clinical examination, when measuring the laccase inhibitor with a biocomponent oxidase-peroxidase-chromogen system, a bilirubin oxidase is used to avoid or reduce interference of bilirubin in the biocomponent. When added to the reaction system used, it prevents the laccase action of bilirubin oxidase.

Various synthetic agents have been conventionally used as laccase inhibitors for the use of herbicides, anti-browning agents and skin cosmetics, and kojic acid and the like have been used as natural substances. It has been desired to develop a substance that can be expected to have the same action by other natural ingredients.

[0005] In the field of clinical examination, interference of bilirubin in measuring biological components by oxidase-peroxidase-chromogen system has been an unsolved problem.

The interference mechanism of bilirubin in the above measurement system mainly includes the following. (1) Direct influence of specific absorption of bilirubin (around 450 nm) (2) Bilirubin becomes a hydrogen donor of peroxidase. (3) The bilirubin acts on the formed coloring pigment to decompose the coloring pigment.

The concentration of bilirubin in serum is 1 mg / dl or less in a normal person, but 20 mg / dl depending on the disease.
However, how to avoid the effect is an important issue in clinical examination.

[0008]

BACKGROUND ART Phosphates and sulfites have been used to prevent browning of cut vegetables, but their use is prohibited due to food hygiene problems, and ascorbic acid and kojic acid have been used for the same purpose. However, the effect was not sufficient (JP-A-2-31661, JP-A-2-69156, JP-A-2-273140).

As the cosmetics, hydroquinones and the like have been used for thinning freckles and the like generated on the surface of the skin, but there are problems with their side effects and it has been difficult to apply them to the cosmetics. Further, there have been reported examples of using kojic acid alone or by mixing other components, but the effect was not satisfactory (JP-A-1-12120).
5, JP-A-3-11010, JP-A-2-15017).

As a method for avoiding interference of bilirubin in the field of clinical examination, for example, a method of adding ferricyanide, ascorbic acid or EDTA-iron complex, or a bilirubin-specific fungal enzyme of mushroom origin or bilirubin oxidase is used as a reaction system. To remove bilirubin (Japanese Patent Publication No. 55-25840, Japanese Patent Laid-Open No. 5-40840).
No. 5,138,656, JP-A-55-29718, JP-A-57-71398, JP-A-59-18000) and the like are known, but all have drawbacks and are not sufficiently satisfactory. I couldn't say it.

[0011]

DISCLOSURE OF THE INVENTION The present invention broadly searches for substances having a laccase inhibitory activity from the natural world and finds that a strain belonging to the genus Penicillium produces the above-mentioned substance of interest. Was identified, it was completed by discovering that it is hadasidin and N-hydroxyglycine, which is a hydrolyzate of hadasidin.

Hadacidin is known as an anticancer antibiotic represented by the chemical formula C ₃ H ₅ NO ₄ (molecular weight 119.08), and inhibits adenylosuccinate synthetase competitively with respect to the substrate L-aspartate, and adenine It is known to have an action of suppressing the biosynthesis of nucleotides. However, it is not known that this substance inhibits the action of laccase. In addition, it was not known at all that N-hydroxyglycine, which is a hydrolyzate of this substance, also has this action.

That is, the feature of the present invention is a laccase inhibitor containing hadacidin or N-hydroxyglycine as an active ingredient, which is applied to herbicides, browning inhibitors, cosmetics and reagents for clinical examination. It is in the place of providing.

More specifically, it can be considered to be used in combination with bilirubin oxidase as a field of application of a clinical test reagent.

Bilirubin oxidase acts on bilirubin to change it to an almost colorless product via biliverdin, and as a result, the specific absorption of bilirubin is reduced and the reducing property of bilirubin is lost. Furthermore, this enzyme has a characteristic that it does not generate hydrogen peroxide, which makes it a particularly useful method for removing interference of bilirubin.

However, bilirubin oxidase is contained in the reagents of the measuring system, 4-aminoantipyrine, phenol, N, N-diethylaniline, N-ethyl-N-.
Since it has a slight reactivity with (2-hydroxy-3-sulfopropyl) -m-toluidine (hereinafter referred to as TOOS) and the like, it has a drawback of increasing the blank value.

The present invention also provides a technique for improving such drawbacks, avoiding or reducing the interference of bilirubin by bilirubin oxidase, and improving the measurement accuracy of clinical tests.

[0018]

Means for Solving the Problems As a result of intensive studies conducted by the present inventors to solve the above problems and achieve the object, it was found that hadacidin and N-hydroxyglycine strongly inhibit laccase. I found it.

The herbicides, browning preventives and cosmetics in the present invention will be described first. In the present invention, hadacidin and / or N-hydroxyglycine are the main active ingredients,
Various kinds of conventionally used drugs may be used in combination therewith.

The amount of the herbicide, browning preventive, or cosmetics can be arbitrarily selected, but is usually 0.001 to 10%.
Is blended.

Next, application to a clinical test reagent will be described. The inventors of the present invention have conducted extensive studies to inhibit the reaction of hadalidine and / or N-hydroxyglycine with bilirubin oxidase to chromogens such as 4-aminoantipyrine, phenol, N, N-diethylaniline and TOOS. However, it was found that the action on bilirubin is hardly inhibited, and by allowing this substance to coexist with bilirubin oxidase for the measurement of oxidase-peroxidase-chromogenic system, bilirubin oxidase eliminates bilirubin to avoid the interference action. The present invention has been completed by discovering that the effect of bilirubin oxidase on the measurement system can be avoided at the same time. In particular, it was found that N-hydroxyglycine has a strong action.

That is, the gist of the present invention is to measure the biological components contained in a sample, especially in a sample such as serum, plasma, urine, etc. used in clinical tests by oxidase-peroxidase-chromogenic system, in which bilirubin oxidase, N -A method for measuring biological components by adding hydroxyglycine to eliminate bilirubin, and this method avoids or reduces interference of bilirubin.

In the present invention, hadacidin is a strain belonging to the genus Penicillium separated from the soil by the present inventors [this strain is Penicillium sp. YH-31 (Peni
Cillium SP. YH-31), and the Institute of Microbial Science and Technology, the Institute of Industrial Science and Technology, has a 1244
Deposited as No. 5 (FERM P-12445). ] (Hereinafter referred to as YH-31 strain) was produced, and further hydrolyzed to produce N-hydroxyglycine.

Hadacidin can be produced by culturing and purifying YH-31 strain under the conditions of Test Example 1, and N-hydroxyglycine can be prepared according to Test Example 2.

Further, hadacidin and N-hydroxyglycine can be produced by the synthetic method as in Test Example 3.

Hadacidin and N-hydroxyglycine inhibit the reaction activity of bilirubin oxidase against chromogens such as 4-aminoantipyrine, phenol, N, N-diethylaniline and TOOS, but hardly inhibit the activity against bilirubin. Have.

Bilirubin oxidase is a genus Myrothecium, Trichoderma (Tri-derma).
genus choderma or Coprinus
s) An enzyme produced by a strain belonging to the genus.

The measurement of oxidase-peroxidase-chromogen in the present invention utilizes an oxidase that produces hydrogen peroxide from the component to be quantified. For example, measurement of glucose using glucose oxidase,
Examples include measurement of cholesterol using cholesterol oxidase, measurement of galactose using galactose oxidase, measurement of uric acid using uricase, measurement of spermidine using amine oxidase, and the like.

As a chromogen, a combination of 4-aminoantipyrine and phenol is mainly used. In addition, in order to increase sensitivity, a combination of 3-methyl-2-benzothiazolinone hydrazone and N, N-dimethylaniline, a combination of 4-aminoantipyrine and p-chlorophenol, a combination of 4-aminoantipyrine and TOOS, 4 -Aminoantipyrine and 3-methyl-N-
(Β-hydroxyethyl) -aniline combination, 4
Many chromogens have come to be used, such as a combination of -aminoantipyrine and N-ethyl-N- (3-methylphenyl) -N'-acetylethylenediamine. Moreover, these can be used even if the combination is changed appropriately.

The present invention will be described in detail below with reference to test examples and examples. Test Example 1 Culture of YH-31 strain and purification of hadasidin Medium composition Meat extract 1% NaCl 0.5% Adecanol 0.01% Peptone 1% Glucose 1% pH 7.2

Cultivation conditions Cultivation temperature 30 ° C. stirring 200 rpm aeration 20 l (1 vvm) 20 l medium / 30 l jar famentor

Purification method To 20 liters of the culture filtrate adjusted to pH 7.0 with 2N sodium hydroxide, 200 g of activated carbon was added and stirred for 2 hours to adsorb impurities on the activated carbon. Filter paper (TOYO filter paper N
o. After filtering off the activated carbon using 2), deionized water 1
It was washed twice with 1 and the washing solution was also added to the filtrate. 1 of this filtrate
The solution was concentrated under reduced pressure to 1 and lyophilized. The dried product was extracted 3 times with 1 liter of methanol, the methanol was distilled off under reduced pressure, the residue was dissolved in 200 ml of water, and 500 ml of ethanol was added for crystallization to obtain 9.9 g of crude crystals. Crude crystal 9.9
g was dissolved in 100 ml of water, 1.0 g of activated carbon for decolorization was added, and the mixture was stirred for 1 hour and then filtered through a filter paper. 300 ml of ethanol was added to the filtrate and recrystallization was performed to obtain 7.8 g of hadadidine Na salt.

Test Example 2 Preparation of N-hydroxyglycine 65 g of 6.5 g of the sodium salt of hadacidin obtained in Test Example 1
After dissolving in ml of water and passing through a cation exchange resin (H ⁺ type) packed in a 100 ml column, 200 ml of water was further poured to wash. The cation exchange resin passing solution and the washing solution were combined and concentrated under reduced pressure to 50 ml, 1.0 ml of trifluoroacetic acid was added, and the mixture was heated in a boiling water bath for 8 minutes. This solution was dried under reduced pressure, washed 3 times with 10 ml of water, added with 35 ml of water, dissolved by heating, and allowed to stand at room temperature to give 1.1 g.
N-hydroxyglycine was obtained.

Test Example 3 Synthesis of hadacidin and N-hydroxyglycine Synthesis of glyoxylic acid oxime Hydroxylamine + glyoxylic acid → glyoxylic acid oxime H ₂ NOH.HCL OHC-COOH.H ₂ O HO-N = CH-COOH

Hydroxylamine hydrochloride (7 g) and glyoxylic acid monohydrate (9.2 g) were dissolved in water (25 ml), and the mixture was stirred overnight at room temperature and extracted with 25 ml of ether. The ether extract was dehydrated with anhydrous magnesium sulfate, filtered with a filter paper, dried under reduced pressure, and recrystallized with ether to obtain 7.1 g of glyoxylic acid oxime.

Synthesis of N-hydroxyglycine Glyoxylic acid oxime Sodium cyanoborohydride N-Hydroxyglycine HO-N = CH-COOH ------------- → HO-NH-CH ₂ -COOH NaBH ₃ CN

1.05 g of sodium cyanoborohydride and 2.23 g of glyoxylic acid oxime were dissolved in 50 ml of methanol, adjusted to pH 2 or less with hydrochloric acid, stirred at room temperature for 1 hour, and dried under reduced pressure. After 20 ml of water was added and the mixture was heated gently until no gas came out, it was cooled and crystallized. The crystals were recrystallized from water to obtain 0.34 g of N-hydroxyglycine.

Synthesis of hadacidin N-hydroxyglycine + formic acid → hadasidin HO—NH—CH ₂ —COOH HCOOH OHC—N (OH) —CH ₂ —COOH

To 91 mg of N-hydroxyglycine, 1.7 ml of formic acid and 0.4 ml of acetic anhydride were added, and 50
It concentrated under reduced pressure which heated at 10 degreeC. The residue was dissolved in 2 ml of water and adjusted to pH 7 with 2N sodium hydroxide,
92 mg of hadacidin was obtained by concentrating under reduced pressure and recrystallizing from a water-ethanol system.

Test Example 4 Properties of hadacidin obtained in Test Example 1 and N-hydroxyglycine obtained in Test Example 2

Hadacidin Na salt N-Hydroxyglycine ------------------------------- Molecular weight (mass spectrum) (Free 119) 91 Elemental analysis C ₃ H ₄ NO ₄ Na C ₂ H ₅ NO ₃ Theoretical value: C: 25.54, H: 2.86, N: 9.92, Na: 16.3 C: 26.37, H: 5.53, N: 15.38 Analysis Value: C: 25.36, H: 2.75, N: 9.87, Na: 16.1 C: 26.03, H: 5.40, N: 15.00 Optical rotation [α] _D (27 ℃) 0 ° (c = 1.00, H ₂ 0) 0 _{° (c = 0.50, H 2} 0) ultraviolet absorption Suhe ° vector terminus absorbing terminal absorption mp 198~199 ℃ (dec.) 141~142 ℃ (dec.) solubility water soluble insoluble under methanol soluble insoluble acetone insoluble insoluble coloring reagent Ninhito Bu phosphate + ＋ Ferric chloride ＋ －

The infrared absorption spectrum and each magnetic resonance spectrum were in agreement with those of hadacidin and N-hydroxyglycine synthesized in Test Example 3 .

Test Example 5 Examination of bilirubin oxidase inhibitory action of N-hydroxyglycine 100 μl of bilirubin oxidase (2.5 μg) and N
-Hydroxyglycine (50-100 μg) 100 μl
After incubation at 37 ° C for 10 minutes, 20 μg / m
0.1 M Tris-hydrochloric acid buffer solution containing 1 bilirubin (pH
2.5 ml of 8.4) is added and reacted at 37 ° C. Four
Enzyme activity was determined by measuring the decrease in absorbance at 40 nm.

The inhibitory activity was determined using the enzyme activity as a control when water was used instead of N-hydroxyglycine.

Test Example 6 Bilirubin oxidase N,
Examination of activity inhibitory action against N-diethylaniline Bilirubin oxidase (50 μg) 100 μl and N-
After incubating 100 μl of hydroxyglycine (25 to 100 μg) at 37 ° C. for 10 minutes, 4 mM N, N
-0.1 M phosphate buffer containing diethylaniline (pH
2.5 ml of 6.0) is added and reacted at 37 ° C. Four
Enzyme activity was determined by measuring the increase in absorbance at 72 nm.

The inhibition rate was determined using the enzyme activity as a control when water was used instead of N-hydroxyglycine.

Test Example 7 4-of bilirubin oxidase
Examination of activity inhibitory action against aminoantipyrine-phenol Bilirubin oxidase (100 μg) 100 μl and N
-Hydroxyglycine (25-100 μg) 100 μl
After incubation at 37 ° C. for 10 minutes, 2.5 ml of 0.1 M phosphate buffer (pH 6.8) containing 2.5 mM 4-aminoantipyrine and 0.17 mM phenol.
Add and react at 37 ° C. Enzyme activity was determined by measuring the increase in absorbance at 510 nm.

The inhibition rate was determined using the enzyme activity as a control when water was used instead of N-hydroxyglycine.

Test Example 8 4-of bilirubin oxidase
Examination of activity inhibitory action against aminoantipyrine-TOOS Bilirubin oxidase (50 μg) 100 μl and N-
After incubating 100 μl of hydroxyglycine (25 to 100 μg) at 37 ° C. for 10 minutes, 0.3 mM of 4
-Aminoantipyrine and 2.5 ml of 0.1 M phosphate buffer (pH 6.8) containing 1.0 mM TOOS are added and reacted at 37 ° C. Enzyme activity was determined by measuring the increase in absorbance at 555 nm.

The inhibition rate was determined by using the enzyme activity as a control when water was used instead of N-hydroxyglycine.

Test Example 9 4-of bilirubin oxidase
Examination of activity inhibitory action against aminoantipyrine-N, N-diethylaniline Bilirubin oxidase (50 μg) 100 μl and N-
After incubating 100 μl of hydroxyglycine (25 to 100 μg) at 37 ° C. for 10 minutes, 0.3 mM of 4
-Aminoantipyrine and 2.5 ml of 0.1 M phosphate buffer (pH 6.8) containing 1.0 mM N, N-diethylaniline are added and reacted at 37 ° C. Enzyme activity was determined by measuring the increase in absorbance at 550 nm.

The inhibition rate was determined using the enzyme activity as a control when water was used instead of N-hydroxyglycine. The results of Test Example 5 to Test Example 9 are shown in Table 1.

[0053]

[Table 1]

From the above results, N-hydroxyglycine hardly inhibits the activity of bilirubin oxidase against bilirubin, but N, N-diethylaniline, 4
-Combination of aminoantipyrine and phenol, 4-
It can be seen that the reaction activity against the combination of aminoantipyrine and TOOS and the combination of 4-aminoantipyrine and N, N-diethylaniline is strongly inhibited.

In the following examples, hadacidin, N-
As the hydroxyglycine, those purified from the HY-31 strain were used, and the others were those specified by the Food Addition Standard.

[0056]

Example 1 Mortality test of wormwood 30 wormwood seedlings were transplanted into pots at a height of about 10 cm (4 to 5 leaves), and were bred for one week as they were. After 1 week, 10 plants were divided into 3 groups so that the growth condition was averaged. Water (target area) for each group, 0.025 wt% aqueous solution of hadacidin,
A 0.025 wt% N-hydroxyglycine aqueous solution was sprayed to such an extent that the surface of the leaf became wet, and the state of the leaf was visually observed. Table 2 shows the changes over time.

[0057]

[Table 2]

The symbols in the table are shown below. −: No change ±: Leaf slightly yellowed +: Leaf wilted ++: Leaf withered

As shown in Table 2, in the group treated with hadacidin and N-hydroxyglycine, the effect of dying wormwood leaves was observed as compared with the group treated.

Example 2 Prevention of Browning of Potatoes Commercially available potatoes were washed, peeled and sliced, and 1
It was divided into 3 groups of 00 g each. Water (control),
After water containing 0.3 wt% of hadacidin, 0.3 wt% of N-
It was immersed in an aqueous solution of hydroxyglycine for 1 minute, lightly drained, placed on a plastic tray, covered with wrap, and allowed to stand at room temperature (20 ° C.), and the state of browning was visually observed. The browning over time is shown in Table 3.

[0061]

[Table 3]

The symbols in the table are shown according to the following criteria. -: No change (browning) ±: Slightly browning +: Browning ++: Significant browning

As shown in Table 3, the effect of preventing the browning of potatoes was recognized in the treated groups of hadacidin and N-hydroxyglycine as compared with the control group.

Example 2 Browning of Apples Commercially available apples were peeled and then sliced, and 100 g each was divided into 3 groups. Each of them was treated in the same manner as in Example 1 with water (control), a 0.3 wt% hadadidine aqueous solution, and 0.3 wt%
Immersed in N-hydroxyglycine aqueous solution for 1 minute, lightly drained, placed on a plastic tray and covered with wrap,
It was left to stand at room temperature (20 ° C.) and the state of browning was visually observed.
The browning over time is shown in Table 4.

[0065]

[Table 4]

The symbols in the table are shown according to the following criteria. -: No change (browning) ±: Slightly browning +: Browning ++: Significant browning

As shown in Table 4, the effect of preventing the browning of apples was observed in the hadadizin and N-hydroxyglycine treatment groups as compared with the control group.

Example 3 Prevention of browning of burdock Commercially available burdock was washed, peeled, and quickly sliced, and divided into 3 groups of 100 g each. Each was treated with water (control), a 0.3 wt% aqueous solution of hadacidine, and a 0.3 wt% aqueous solution of N-hydroxyglycine in the same manner as in Example 1.
After dipping for a minute, lightly draining, placing it on a plastic tray, covering with a plastic wrap, and allowing it to stand at room temperature (20 ° C.), the state of browning was visually observed. The browning over time is shown in Table 5.

[0069]

[Table 5]

The symbols in the table are shown according to the following criteria. -: No change (browning) ±: Slightly browning +: Browning ++: Significant browning

As shown in Table 5, in the N-hydroxyglycine treated group, the effect of preventing browning of burdock was observed as compared with the target group.

Example 4 Prevention of browning of yam Yam on the market was washed, peeled and grated with a grater, and 20 g each was divided into 5 groups. To each of them, 0.5 ml of each of the solutions (A) to (D) shown in Table 6 was added, and 0.5 ml of water was added to the untreated section (target section). Cover, room temperature (20
It was left to stand at (° C.) and the appearance of browning was visually observed. In addition, ascorbic acid was added as an enhancer for preventing browning. The browning over time is shown in Table 7.

[0073]

[Table 6]

[0074]

[Table 7]

The symbols in the table are shown according to the following criteria. -: No change (browning) ±: Slightly browning +: Browning ++: Significant browning

As shown in Table 7, in the treated section (C), the effect of preventing browning of the yam was recognized as compared with the target section.

Example 5 Prevention of Browning of Miso A mixture of hadasidin or N-hydroxyglycine and commercially available seed koji was added to steamed rice, and koji was made at 28 ° C. for 3 days. Then, the mixture was placed in a 20-liter beaker together with salt and steamed soybeans and aged at room temperature (20 ° C.) for 2 months. Then, take a portion of the aged miso from the center and use the colorimeter ND-1
The color was measured by using 001DP (manufactured by Nippon Denshoku Co., Ltd.). The results are shown in Table 8.

[0078]

[Table 8]

As shown in Table 8, it was confirmed that, in the groups containing hadacidin and N-hydroxyglycine, the browning of the miso was suppressed in comparison with the control group.

Example 6 Sun Exposure Test Five healthy males were provided with four sites of 5 × 5 cm each on the upper back and forearm. 50 mg of each of the sample A, the sample B, and the sample C shown in Table 9 was uniformly applied to each site. Further, 50 ml of water was applied to the target area. Each part of the upper back and forearm was exposed to direct sunlight for 4 hours, and the appearance of red spots was visually observed after 4 hours and 1 day. The results are shown in Table 10 (after 4 hours) and Table 11 (after 1 day).

[0081]

[Table 9]

[0082]

[Table 10]

[0083]

[Table 11]

The symbols in the table indicate the number of people according to the following criteria. -: No change (red spot) ±: Slight red spot +: Red spot ++: Very intense red spot

As shown in Tables 10 and 11, in comparison with the control group and the control (Sample A), the group containing hadacidin (Sample B) and the group containing N-hydroxylysin (Sample C) showed the occurrence of red plaque. A suppressive effect was observed.

Example 7 0.1 ml of each of the following samples (1) to (4) was taken in a test tube, and then the following reagent A was added and incubated at 37 ° C. for 5 minutes. Then, the reagent B was added and reacted at 37 ° C. for 5 minutes, and the absorbance at 555 nm was measured. Similarly, the measurement was performed using a reagent system containing no bilirubin oxidase and N-hydroxyglycine.

Sample 20 mg / dl containing albumin-bound bilirubin 1
5 mg / dl glucose solution 10 mg / dl containing albumin-bound bilirubin 1
5 mg / dl glucose solution 15 mg / dl glucose solution

Reagent A 0.2 M phosphate buffer (pH 7.0) 0.5 ml (containing 1.8 mM 4-aminoantipyrine) 200 mg / ml sodium cholate 0.1 ml 8 mg / ml N-hydroxyglycine 0.5 ml bilirubin Oxidase (3 units / ml) 0.1 ml (Mirocesium origin)

Reagent B 0.1 M phosphate buffer (pH 7.0) 2.0 ml [TOOS (1.5 mM), glucose oxidase (30 units / ml) Peroxidase (10 units / ml) Mutarotase (1 unit / ml) Content] The results are shown in Table 12.

[0090]

[Table 12]

It can be seen that the bilirubin oxidase and the N-hydroxyglycine-free reagent system are affected by bilirubin, but the bilirubin oxidase- and N-hydroxyglycine-containing reagent system avoids the bilirubin interference.

Example 8 The same procedure as in Example 7 was carried out by using bilirubin oxidase derived from Trichoderma genus instead of bilirubin oxidase derived from genus Milocesium. As a result, the real
Results similar to those in Example 7 were obtained.

Example 9 Samples (1) to (4) used in Example 7 were added to test tubes at 0.
After collecting 1 ml, the following reagent A was added and incubated at 37 ° C. for 5 minutes. Then add reagent B and add 37
The reaction was carried out at 5 ° C for 5 minutes, and the absorbance at 550 nm was measured.

Similarly, the measurement was carried out using a reagent system containing neither bilirubin oxidase nor N-hydroxyglycine. Reagent A 0.2 M phosphate buffer (pH 7.0) 0.5 ml (containing 1.8 mM 4-aminoantipyrine) 200 mg / ml sodium cholate 0.1 ml 0.8 mg / ml N-hydroxyglycine 0.5 ml Bilirubin oxidase (3 units / ml) 0.1 ml (Mirocesium origin)

Reagent B 0.1 M phosphate buffer (pH 7.0) 2.0 ml [TOOS (1.5 mM), sodium azide (5 mM) glucose oxidase (30 units / ml) peroxidase (10 units / ml) mutarotase (1 unit / ml) included] The results are shown in Table 13.

[0096]

[Table 13]

It can be seen that the bilirubin interference was avoided in the reagent system containing bilirubin oxidase and N-hydroxyglycine.

Example 10 0.1 ml of each of the following samples (1) to (4) was taken in a test tube, and then the following reagent A was added and incubated at 37 ° C. for 5 minutes. Then, the reagent B was added and reacted at 37 ° C. for 5 minutes, and the absorbance at 550 nm was measured. Similarly, the measurement was performed using a reagent system containing no bilirubin oxidase and N-hydroxyglycine.

Sample 5 mg containing 20 mg / dl albumin-bound bilirubin
0 mg / dl cholesterol solution 10 mg / dl containing albumin-bound bilirubin 5
0 mg / dl cholesterol solution 50 mg / dl cholesterol solution

Reagent A 0.2 M phosphate buffer (pH 7.0) 0.5 ml (containing 1.8 mM 4-aminoantipyrine) 200 mg / ml sodium cholate 0.1 ml 8 mg / ml N-hydroxyglycine 0.1. 5 ml Bilirubin oxidase (3 units / ml) 0.1 ml (Mirocesium origin)

Reagent B 0.1 M phosphate buffer (pH 7.0) 2.0 ml [phenol (0.09%), cholesterol oxidase (1.5 units / ml), peroxidase (10 units / ml), Triton X -100 (0.1%) content] The results are shown in Table 14.

[0102]

[Table 14]

It can be seen that the reagent system containing no bilirubin oxidase and N-hydroxyglycine is affected by bilirubin, while the reagent system containing bilirubin oxidase and N-hydroxyglycine avoids the interference of bilirubin.

Example 11 The same procedure as in Example 10 was carried out by using bilirubin oxidase derived from Trichoderma genus in place of bilirubin oxidase derived from genus Milocesium. as a result,
Results similar to those in Example 10 were obtained.

[0105]

According to the present invention, hadacidin and / or N
-Hydroxyglycine can be applied to herbicides, anti-browning agents, cosmetics and the like, and N-hydroxyglycine is 4-aminoantipyrine of bilirubin oxidase, phenol,
Utilizing the fact that it inhibits the reaction to N, N-diethylaniline or TOOS, but hardly the action to bilirubin, this substance is used as an oxidase-peroxidase-
By allowing bilirubin oxidase to coexist with bilirubin oxidase in the measurement of the chromogenic system, bilirubin oxidase can eliminate bilirubin to avoid an interference effect, and at the same time, the influence of bilirubin oxidase on the measurement system can be avoided.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12Q 1/26 6807-4B 1/28 6807-4B

Claims (8)

[Claims] 1. A laccase inhibitor containing hadacidin and / or N-hydroxyglycine as an active ingredient. 2. A herbicide containing, as an active ingredient, hadacidin and / or N-hydroxyglycine. 3. An agent for preventing browning of foods, which comprises hadacidin and / or N-hydroxyglycine as an active ingredient. 4. A cosmetic comprising, as an active ingredient, hadacidin and / or N-hydroxyglycine. 5. A laccase action inhibitor of bilirubin oxidase containing N-hydroxyglycine as an active ingredient. 6. A method for inhibiting the laccase action of bilirubin oxidase by N-hydroxyglycine. 7. A method for eliminating bilirubin by bilirubin oxidase, which comprises coexisting N-hydroxyglycine. 8. A bilirubin oxidase and N- in a measuring method using an oxidase, a peroxidase and a chromogen.
A method for removing interference of bilirubin, which comprises coexisting with hydroxyglycine.