WO2020116546A1

WO2020116546A1 - Bilirubin oxidase storage method, bilirubin oxidase product, and hair dye

Info

Publication number: WO2020116546A1
Application number: PCT/JP2019/047550
Authority: WO
Inventors: 悠加戸; 山田　浩之; 宏和坪井; 隆之坊垣; 弘雅仙波; 明生幸田; 一俊橋本; 萌植田; 義雄辻野
Original assignee: 大関株式会社
Priority date: 2018-12-05
Filing date: 2019-12-05
Publication date: 2020-06-11
Also published as: JP2022024205A

Abstract

Provided is a novel bilirubin oxidase (BOD) storage method, whereby it becomes possible to prevent an oxidation reaction with BOD from being inhibited and to store BOD stably. The BOD storage method according to the present invention is a method comprising storing a BOD solution containing BOD and a specific stabilizing agent under a deoxidizing atmosphere.

Description

Method for storing bilirubin oxidase, bilirubin oxidase product, and hair dye

The present invention relates to a stable storage method for bilirubin oxidase. The present invention also relates to a bilirubin oxidase product that is stable for a long period of time and does not inhibit the oxidation reaction of bilirubin oxidase, and a hair dye containing the bilirubin oxidase product.

Bilirubin oxidase (hereinafter abbreviated as “BOD” as appropriate) is an enzyme that catalyzes a reaction that oxidizes bilirubin to biliverdin. BOD is used for measuring the concentration of bilirubin in a biological sample, for example, in the field of clinical examination. Further, since BOD has a property of oxidizing an indole analog, it is expected to be applied to a hair dye.

Commercially supplied BOD products include, in addition to BOD, (a) a buffer that gives a suitable pH for an enzymatic reaction, and (b) (b-1) a surfactant, etc., if necessary. And (b-2) common excipients such as sugars and/or inactive proteins, and the like. Currently commercially available BOD products are distributed in freeze-dried form with the above-mentioned components in addition to BOD. The BOD in a dried state is used by dissolving it in a predetermined solvent containing a surfactant and/or a buffer at the time of use. The BOD contained in the product is relatively stable in a dry state, but has a problem that it is easily deactivated in a solution state once dissolved. For example, the commercially available BOD reagent is only 96 hours stable when stored at 5° C. in Tris-H ₂ SO ₄ (pH 8.5) buffer.

In addition, since dry BOD requires an operation to dissolve the required amount each time it is used, the number of steps in use increases, and the BOD powder to the operator is scattered due to scattering of BOD powder during use. There are also problems such as exposure. In addition, it is desirable that the BOD product be distributed at room temperature from the viewpoint of distribution cost.

Considering the above circumstances, a method for stably storing BOD of BOD products (particularly in solution) is being studied. For example, Patent Document 1 discloses a method for stabilizing BOD using a reducing agent (sodium thiosulfate).

Japanese Patent Laid-Open No. 10-42869 (Published February 17, 1998)

However, the conventional technique as described above has a problem that the reducing agent (sodium thiosulfate) used inhibits the oxidation reaction by BOD. One embodiment of the present invention has been made in view of the above problems, and an object thereof is to store a novel BOD that can stably store the BOD without inhibiting the oxidation reaction by the BOD. It is to provide a method.

Another embodiment of the present invention aims to provide a BOD product that is stable for a long period of time and does not inhibit the oxidation reaction of BOD, and a hair dye containing the BOD product.

As a result of intensive studies to solve the above problems, the inventors of the present invention stored the BOD solution containing BOD and a specific substance (a “stabilizer” described later) in a deoxygenated atmosphere, thereby solving the above-mentioned problems. The present invention has been completed and the present invention has been completed.

That is, one embodiment of the present invention is a method for preserving bilirubin oxidase, which comprises storing a bilirubin oxidase solution containing bilirubin oxidase and a stabilizer under a deoxygenated atmosphere: The agent has an ABTS initial absorbance ( _A436 ) reduction rate (%) of 0.10% or more, and a DMBQ initial absorbance ( _A256 ) reduction rate (%) of 0.80% or less.

In another embodiment of the present invention, the container contains a bilirubin oxidase and a stabilizer, the container has a deoxygenated atmosphere, and the stabilizer has an ABTS initial absorption (A ₄₃₆ ) reduction rate. (%) is 0.10% or more, and the DMBQ initial absorbance (A ₂₅₆ ) reduction rate (%) is 0.80% or less, which is a bilirubin oxidase product.

Another embodiment of the present invention is a hair dye, which comprises the bilirubin oxidase product according to one embodiment of the present invention.

According to one embodiment of the present invention, it is possible to stably store BOD without inhibiting the oxidation reaction by BOD.

Further, surprisingly, according to one embodiment of the present invention, the effect that the specific activity of BOD is improved as compared with that before storage was obtained. This is an advantageous effect that one skilled in the art would not have expected according to an embodiment of the present invention.

In addition, according to the BOD product according to one embodiment of the present invention, it is stable for a long time and does not inhibit the oxidation reaction of BOD, so that a hair dye containing the BOD product can be provided.

(A) and (b) show the residual activity of BOD as a relative value to the control when BOD was stored using a storage method outside the scope of the present invention or a storage method according to an embodiment of the present invention. FIG. 6C is a graph, and FIG. 6C is a table showing residual activity of BOD when BOD is stored using a storage method according to an embodiment of the present invention or a storage method outside the scope of the present invention. It is a figure which shows ABTS initial absorption ( _A436 ) reduction rate (%) by the stabilizer or the comparative substance which concerns on one Embodiment of this invention. It is a figure which shows the DMBQ initial absorption ( _A256 ) reduction rate (%) by the stabilizer or comparative substance which concerns on one Embodiment of this invention. It is a figure which shows the result of having performed the hair dye test by the hair dye which concerns on one Embodiment of this invention, or the hair dye outside the scope of this invention.

An embodiment of the present invention will be described below, but the present invention is not limited to this. The present invention is not limited to each configuration described below, and various modifications can be made within the scope of the claims. That is, embodiments or examples obtained by combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, new technical features can be formed by combining the technical means disclosed in each of the embodiments. Moreover, all the patent documents described in this specification are used as a reference in this specification. Unless otherwise specified in the present specification, “A to B” representing a numerical range means “A or more (including A and larger than A) and B or less (including B and smaller than B)”.

In the present specification, X according to one embodiment of the present invention (X is an arbitrary proper noun, for example, preservation method, enzyme solution, BOD, stabilizer, etc.) may be simply referred to as “the present X”. ..

[1. Technical idea of one embodiment of the present invention]
Conventionally, the cause of inactivation of BOD has been considered to be due to oxidation, and a reducing agent has been used to stably store BOD. As the reducing agent, a reducing agent having a relatively strong reducing power tends to be preferably used.

BOD can be used for various purposes other than the purpose of measuring the concentration of bilirubin. An example of the use of BOD is as an oxidation catalyst contained in an oxidation dye for dyeing keratin fibers such as hair. The use of BOD as an oxidation catalyst utilizes the property that BOD oxidizes indole analogs. The present inventors have found for the first time that the following problems occur when BOD stored by a conventional method (that is, BOD containing a reducing agent) is used as an oxidation catalyst for an oxidation dye. That is, there is a problem that the reducing agent added to the oxidative dye together with BOD inhibits the oxidation of BOD with respect to the indole analog, and as a result, the hair dyeability of the oxidative dye. The present inventors diligently studied to solve such a problem. As a result, the inventors of the present invention used a substance whose ABTS initial absorption ( _A436 ) reduction rate (%) and DMBQ initial absorption ( _A256 ) reduction rate (%) were within a specific range, and deoxidized. The present inventors have found a new finding that BOD can be stably stored under an atmosphere, and completed the present invention.

[2. Preservation method〕
This storage method is a method of storing a BOD solution containing BOD and a stabilizer in a deoxygenated atmosphere. Here, the above stabilizer has an ABTS initial absorption ( _A436 ) reduction rate (%) of 0.10% or more, and a DMBQ initial absorption ( _A256 ) reduction rate (%) of 0.80% or less. Is.

By having the above configuration, this storage method can stably store BOD in a solution state without inhibiting the oxidation reaction catalyzed by BOD. That is, the present storage method has an advantage that the oxidation reaction by BOD is not inhibited even when the BOD solution stored by the present storage method is used for various purposes.

Unless otherwise specified herein, the “BOD solution” means a solution containing at least BOD and a stabilizer. The BOD solution may contain a substance other than BOD and the stabilizer.

In the present specification, “stable storage of BOD” means that a BOD solution containing BOD and a specific substance is stored under a deoxygenated atmosphere in a storage of the BOD solution containing a specific substance under a deoxidized atmosphere. It refers to storing BOD in a state in which the activity of BOD is kept higher than when it was stored. In the present specification, the specific substance is referred to as “stabilizer”. In addition, the activity of BOD is often represented by the activity (activity/mass) with respect to the mass of the BOD or the composition containing the BOD, and thus the activity may be referred to as “specific activity”.

The specific activity can be calculated as follows. The value obtained by dividing the absorbance (A ₂₈₀ ) of the BOD solution containing only BOD at a wavelength of 280 nm by the molecular extinction coefficient of 115,000 was multiplied by the molecular weight of BOD of 66,000, and the obtained value was calculated as the BOD protein concentration in the BOD solution ( mg/mL). The enzyme activity (U/mL) of BOD was measured by the method described below, and the value obtained by dividing the enzyme activity by the BOD protein concentration was the specific activity (U/mg).

In this storage method, it is preferable to store BOD stably for a long period of time. In this storage method, it is preferable to store the BOD for 14 days or more, and to store the BOD for 18 days or more, while maintaining the BOD activity higher than when the BOD solution containing no stabilizer is stored. It is more preferable to store the BOD for 21 days or longer, it is more preferable to store the BOD for 33 days or longer, and it is particularly preferable to store the BOD for 35 days or longer.

In the present storage method, the temperature for storing BOD is not particularly limited, but is preferably 40° C. or lower, more preferably 30° C. or lower, further preferably 20° C. or lower, particularly preferably 10° C. or lower. is there.

In the present specification, the activity of BOD is briefly evaluated as follows. After adding BOD to the solution containing the ABTS substrate, the resulting solution is reacted at 37°C. During the reaction, the BOD activity is evaluated by measuring the increase in the absorbance of the solution at a wavelength of 436 nm with time. A specific method for measuring and evaluating the BOD activity will be described in the test example described later.

(2-1) BOD
As the BOD to which the present preservation method can be applied, (1) all known BODs (non-recombinant BODs) obtained from any organism, and (2) a gene encoding a BOD derived from any organism can be arbitrarily selected. Recombinant BOD obtainable by expression in the organism or in vitro, (3) Mutant BOD obtained by mutation such as substitution, insertion and deletion of amino acid sequence of BOD derived from any organism, and (4 ) BOD modified with any carrier for the purpose of stabilizing BOD and improving the enzymatic activity of BOD, and the like. Examples of the above-mentioned organisms include, for example, genus Milocesium (for example, Milocesium bergaria), Penicillium jancinerum, Bacillus rifeniformis, Corynebacterium glutamicum, Escherichia coli, Agaricus bisporus mushroom, Suehirotake mushroom, edible mushroom, Asteraceae. Plants, alfalfa, Schizosaccharomyces pombe, Pichia pastoris, and Aspergillus, and the like, but are not limited thereto. The organism from which the gene encoding BOD is derived and the organism into which the gene is introduced and expressed to obtain BOD may be the same or different.

The BOD may be (1) a BOD (non-recombinant BOD) obtained from the genus Milocesium, or (2) a mutation obtained by mutation such as substitution, insertion and deletion of the amino acid sequence of the BOD derived from the genus Milocesium. BOD may be used, (3) a gene encoding a BOD derived from the genus Myrocesium may be a recombinant BOD obtained by expressing the gene in any organism or in a test tube, and (4) genus Myrocesium A recombinant BOD obtained by introducing a gene encoding a BOD derived from the codon into the organism after codon optimization according to the gene sequence of the organism and expressing the gene in the organism. Good. For example, the recombinant BOD may be (1) a BOD obtained by introducing a gene encoding a BOD derived from an arbitrary organism into the genus Aspergillus and expressing it in the genus Aspergillus, and (2) optionally A gene encoding a BOD derived from an organism may be a BOD obtained by introducing codons into Aspergillus and then expressing in Aspergillus after codon optimization according to the gene sequence of Aspergillus. .. More specifically, the recombinant BOD is preferably (1) a BOD obtained by introducing a gene encoding a BOD derived from the genus Milocesium into the genus Aspergillus and expressing it in the genus Aspergillus, (2) BOD obtained by introducing a gene encoding a BOD derived from the genus Milocesium into the genus Aspergillus after codon optimization according to the gene sequence of the genus Aspergillus and expressing it in the genus Aspergillus. Is more preferable.

The concentration of BOD used in this storage method is not particularly limited and may be appropriately set depending on the use of the BOD.

(2-2) Stabilizer The present stabilizer is a substance used for stably storing BOD in a deoxygenated atmosphere, and can be said to be a stabilizing aid. This stabilizer has (a) ABTS initial absorbance (A ₄₃₆ ) reduction rate (% = reduction rate of absorbance at wavelength 436 nm per minute) of 0.10% or more, and (b) DMBQ initial absorbance. There is no particular limitation as long as it is a substance having a (A ₂₅₆ ) reduction rate (%=reduction rate of absorbance at a wavelength of 256 nm per minute) of 0.80% or less. As the stabilizer, any compound, mixture, composition or the like can be used.

The stabilizer is preferably at least one substance selected from the group consisting of naturally occurring inorganic salts, carbohydrates, amino acids, fatty acids, lipids, proteins, peptides and the like. The stabilizer may also be any combination of the substances listed in the above group.

Examples of such stabilizers include, but are not limited to, (a) inorganic substances such as uric acid and glyoxylic acid, and (b) peptones (casein peptone, meat peptone, myocardial peptone, gelatin peptone, And soybean peptone), extracts (meat extract, fish extract, plant extract, malt extract, and yeast extract), oligopeptides, corn steep liquor, soybean flour, organic substances such as wheat bran, and the like. One or more substances may be mentioned.

As the above-mentioned inorganic substance, for example, one obtained from Wako Pure Chemical Industries, Ltd. can be used. Examples of the organic matter include malt extract manufactured by Kyokuto Pharmaceutical Co., fish powder extract manufactured by Kyokuto Pharmaceutical Co., powder yeast extract manufactured by Kyokuto Pharmaceutical Co., Far East peptone manufactured by Kyokuto Pharmaceutical Co., and peptone A manufactured by Kyokuto Pharmaceutical Co., Ltd. , Kyokuto Pharmaceutical Co., Ltd. Patty Case, Kyokuto Pharmaceutical Co., Potato Peptone CP, Asahi Group Foods Yeast Extract (for example, Mist P1G and Yeastoc S-Pd), Oriental Yeast Co., Ltd. corn steep liquor (abbreviated as " (Also referred to as "CSL"), Hippolypepton manufactured by Nippon Pharmaceutical Co., Ltd., Hinew AM which is a soybean oligopeptide manufactured by Fuji Oil Co., Soybean Powder 88 manufactured by Showa Sangyo Co., Tryptone manufactured by Japan BD, and Yeast manufactured by BD Japan. Examples include, but are not limited to, at least one or more substances selected from the group consisting of Extract and the like. When the stabilizer is at least one selected from the above group, the stabilizer does not inhibit the oxidation reaction by BOD, and the storage method stabilizes BOD for a long period of time. Can be saved as The various stabilizers listed in the present specification may be used alone or in combination of two or more.

Also, in this storage method, the optimum concentration of the stabilizer contained in the BOD solution may vary depending on the concentration of BOD in the BOD solution, the type of the stabilizer, and the like. Therefore, the final concentration of the stabilizer contained in the BOD solution may be appropriately set within a range in which the effect according to the embodiment of the present invention can be obtained.

(2-3) Deoxygenated Atmosphere In the present preservation method, in order to prevent the oxidation of BOD, it is preferable that the atmosphere in which BOD is present is as free of oxygen as possible (that is, deoxidized atmosphere). Therefore, in the present storage method, the BOD solution containing BOD and the stabilizer is stored under the “deoxygenated atmosphere”. In the present storage method, the “deoxygenated atmosphere” means a state in which the oxygen concentration in the atmosphere in which the BOD solution is stored is lower than the oxygen concentration existing in a natural state (in other words, normal temperature and normal pressure).

In this storage method, by storing the BOD solution in a deoxygenated atmosphere, it is possible to prevent BOD from being oxidized (that is, deactivating the enzyme). Therefore, this storage method has an advantage that BOD can be stored stably.

In this storage method, from the viewpoint of stable storage of BOD, the oxygen concentration in the atmosphere in which the BOD solution is stored is preferably less than 0.5% by volume, particularly preferably less than 0.1% by volume.

In this preservation method, the method for achieving a deoxygenated atmosphere is not particularly limited. For example, in the present storage method, (1) the oxygen scavenger may be achieved by installing the oxygen scavenger in a container for storing the BOD solution, or (2) the oxygen scavenger may be prepared from a material having the oxygen scavenger component A deoxidizing atmosphere may be achieved by storing the BOD solution in a container, or (3) deoxidizing by replacing oxygen in the container with an inert gas (nitrogen gas and a rare gas such as argon). An atmosphere may be achieved, or (4) a deoxygenated atmosphere may be achieved by storing the BOD solution under vacuum or reduced pressure conditions. As a method for achieving these deoxygenated atmospheres, one method may be used alone, or a plurality of methods may be used in combination.

The above method (1) is preferable from the viewpoint of simplicity. Examples of the oxygen scavenger used in (1) above include AGELESS (registered trademark) manufactured by Mitsubishi Gas Chemical Co., Inc., Ageless Omak (registered trademark) manufactured by Mitsubishi Gas Chemical Co., Ltd., Aneropack (registered trademark) manufactured by Mitsubishi Gas Chemical Co., Ltd., Examples include, but not limited to, Everfresh manufactured by Toriyoshi Sangyo Co., Ltd., Wonder Keep (registered trademark) manufactured by Powder Tech Co., Ltd., and the like.

In addition, in the present storage method, from the viewpoint of more stable storage of BOD, in addition to storing the BOD solution containing BOD under a deoxygenated atmosphere, the dissolved oxygen concentration in the BOD solution is set to a low dissolved oxygen state (preferably It is less than 0.2 mg/L, and particularly preferably less than 0.04 mg/L). The dissolved oxygen concentration in the BOD solution is more preferably equal to or lower than the saturated oxygen concentration in the solution under the above-mentioned preferable oxygen concentration in the atmosphere in which the BOD solution is stored. The saturated oxygen concentration in the solution may change depending on the temperature of the solution. For example, when the oxygen concentration in the air atmosphere is 0.5% by volume, the saturated oxygen concentration in the solution is 0.2 mg/L at 25°C and 0.35 mg/L at 0°C. A preferred mode of the dissolved oxygen concentration in the BOD solution (low dissolved oxygen state) is achieved by performing a known degassing operation on the BOD solution.

(2-4) ABTS Initial Absorbance Reduction Rate In the present specification, the absorbance at a wavelength of 436 nm of the solution containing ABTS (ABTS solution) is referred to as the ABTS initial absorbance (A ₄₃₆ ). In the present specification, the ABTS initial absorbance (A ₄₃₆ ) reduction rate (%) of the stabilizer means the ABTS initial absorbance (A ₄₃₆ ) of the absorbance of the mixed solution containing the ABTS solution and the stabilizer at a wavelength of 436 nm. It is defined as the reduction rate (%) per minute with respect to (100%). The ABTS solution is a solution in which the concentration of ABTS is adjusted so that the absorbance (A ₄₃₆ ) at a wavelength of 436 nm is about 1.3 when the optical path length is 1.0 cm. Here, the value "about 1.3" means a value in a range in which the value obtained by rounding off the second decimal place is 1.3. A specific method for measuring the absorbance (A ₄₃₆ ) of the ABTS solution at a wavelength of 436 nm and the ABTS initial absorbance (A ₄₃₆ ) reduction rate (%) of the stabilizer will be described in Test Examples to be described later. ABTS is an abbreviation for 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid).

In the present preservation method, when the ABTS initial absorbance ( _A436 ) reduction rate (%) of the above stabilizer is measured, the concentration of the stabilizer in the ABTS solution for measurement is (a) the stabilizer alone. If the molecular weight of the stabilizer is one mM, it is 1 mM, and (b) if the stabilizer is a substance having a composition of unknown molecular weight, the nitrogen element content is As a standard, the concentration is such that the amount of nitrogen element derived from the stabilizer in 100 mL of the ABTS solution for measurement is 0.011 g. In addition, in (b) above, the nitrogen content derived from the stabilizer in the ABTS solution is a value calculated by the Kjeldahl method.

The present stabilizer may have an ABTS initial absorbance (A ₄₃₆ ) reduction rate (%) of 0.10% or more. The ABTS initial absorbance (A ₄₃₆ ) reduction rate (%) is preferably 0.25% or more, more preferably 0.50% or more, still more preferably 1.00% or more, It is particularly preferably at least 3.00%. According to the said structure, it has the advantage that BOD can be preserved more stably and for a longer period of time.

(2-5) Reduction rate of DMBQ initial absorbance (A ₂₅₆ ) In this specification, the absorbance at a wavelength of 256 nm of a mixed solution immediately after the stabilizer is added to a solution containing DMBQ (DMBQ solution) is referred to as the DMBQ initial absorbance (A _{256 ).} ). In the present specification, the DMBQ initial absorbance (A ₂₅₆ ) reduction rate (%) of the stabilizer means the DMBQ initial absorbance (A ₂₅₆ ) of the absorbance of the mixed solution containing the DMBQ solution and the stabilizer at a wavelength of 256 nm. It is defined as the reduction rate (%) per minute with respect to (100%). The DMBQ solution is a solution in which the concentration of DMBQ is adjusted so that the absorbance (A ₂₅₆ ) at a wavelength of 256 nm is about 1.8 at an optical path length of 1.0 cm. Here, the value "about 1.8" means a value in a range in which the value obtained by rounding off the second decimal place is 1.8. A specific method for measuring the absorbance (A ₂₅₆ ) of the DMBQ solution at a wavelength of 256 nm and the DMBQ initial absorbance (A ₂₅₆ ) reduction rate (%) of the stabilizer will be described in Test Examples below. DMBQ is an abbreviation for 2,5-dimethyl-1,4-benzoquinone.

In the present preservation method, when the DMBQ initial absorbance (A ₂₅₆ ) reduction rate (%) of the above stabilizer is measured, the concentration of the stabilizer in the DMBQ solution for measurement is (a) the stabilizer alone. In the case where the molecular weight of the stabilizer composed of one compound is clear, the concentration is set to 0.1 mM, and when (b) the stabilizer is a substance composed of a composition whose molecular weight is unknown, a nitrogen element is contained. Based on the amount, the concentration is such that the amount of nitrogen element derived from the stabilizer in 100 mL of the DMBQ solution for measurement is 0.0011 g. In addition, in the above (b), the nitrogen content derived from the stabilizer in the DMBQ solution is a value calculated by the same method as in the above (2-4).

The present stabilizer may have a DMBQ initial absorbance (A ₂₅₆ ) reduction rate (%) of 0.80% or less. The DMBQ initial absorbance (A ₂₅₆ ) reduction rate (%) is preferably 0.70% or less, more preferably 0.50% or less, and further preferably 0.20% or less, In the measuring method herein, it is particularly preferably 0.00% or less, that is, the detection limit or less. The above configuration has the advantage of not inhibiting the oxidation reaction catalyzed by BOD.

(2-6) Solvent In this storage method, BOD is contained in the solution together with the stabilizer. The solvent for forming the BOD solution is not particularly limited as long as it can dissolve the present BOD and the present stabilizer, and conventionally known solvents can be used. The present solvent is preferably water (for example, distilled water) from the viewpoint that the present BOD and the present stabilizer can be easily dissolved.

The present BOD solution may have an arbitrary pH depending on the use of the present BOD solution. The present BOD solution preferably has a pH of 6 to 10 in order to keep the three-dimensional structure of BOD contained in the BOD solution stable. The BOD solution preferably contains a buffer to have the desired pH. A conventionally known buffering agent can be used as the buffering agent. Further, since the present BOD solution has a desired pH, the pH can be adjusted by a conventionally known method using a conventionally known acid or alkali.

(2-7) Others The present BOD solution contains any substance other than BOD, a stabilizer, and a solvent as long as it does not inhibit the oxidation reaction by BOD and can stably store BOD. May be included. The present BOD solution may contain, for example, a buffer, an acid, a base, an inorganic salt, an organic salt, a metal salt, a saccharide, an amino acid, an inactive protein, a peptide, a fatty acid, a lipid, a surfactant, and a resin. ..

(2-8) Use The BOD solution stored by this storage method can be used for any purpose. The BOD solution preserved by the present preservation method is, for example, hair dyeing, measurement of the concentration of bilirubin in a biological sample, removal of bilirubin in a biological sample, cosmetics, food processing, enzyme fuel cell (for example, enzyme bio battery, enzyme Batteries, etc.), and protein cross-linking. Since the stabilizer used in the present preservation method does not inhibit the oxidation reaction by BOD, the BOD solution preserved by the present preservation method can be suitably used for the various applications described above, and among the various applications, hair dyeing is possible. Can be more preferably used.

[3. BOD products]
In another embodiment of the present invention, a BOD and a stabilizer are contained in the container, the container is in a deoxygenated atmosphere, and the stabilizer is the ABTS initial absorption (A ₄₃₆ ) reduction rate (%). Is 0.10% or more, and the DMBQ initial absorption (A ₂₅₆ ) reduction rate (%) is 0.80% or less.

The BOD contained in the BOD product has the advantages that it is stable for a long period of time and that the oxidation reaction of the BOD is not hindered by having the above-mentioned configuration.

The BOD and the stabilizer contained in the BOD product may be in a solid state such as powder, but it is preferably a solution dissolved in a suitable solvent (that is, a BOD solution). Although BOD is unstable in a solution state, this BOD product allows stable storage even when the BOD is in a solution state.

When the BOD and the stabilizer contained in the present BOD product are in a solution state, (a) the operation of dissolving the BOD and the activity improver is not required each time it is used, and (b) at the time of use This has the advantage that there is no risk of the BOD being exposed to the worker due to the scattering of the BOD powder.

Note that the description of BOD, stabilizer, solvent, etc. contained in this BOD product is given in the above [2. Storage method] can be incorporated by reference. Further, the BOD product may further contain any substance described in the above (2-7) and other sections. Further, in the present BOD product, the deoxidizing atmosphere in the container can be achieved by the method for forming the deoxidizing atmosphere described in the above section (2-3) Deoxidizing atmosphere.

(3-1) Container As long as the container that can be used in the present BOD product is a container that can store at least BOD and a stabilizer, the material, shape, size, etc. of the container are not limited. The container may be a container that can maintain the deoxidized state in the container for a certain period of time, or may be a container that can create a deoxygenated atmosphere in the container. The material of the container may be glass or resin. The shape of the container may be bottle-like or bag-like. As the bag-shaped container, a resin bag with a fastener (for example, Ziplock (registered trademark)) can be used. The size of the container may be appropriately designed depending on the BOD and the stabilizer to be stored or the volume of the BOD solution.

The container is preferably made of a material that does not permeate oxygen (or a material that does not easily permeate oxygen) in order to maintain the deoxidized state easily and for a long time. Further, the container may be made of a material containing a component having an oxygen scavenger property. Further, the present container is preferably a semi-closed container, and more preferably a closed container that does not allow any substance to permeate, in order to enable stable storage of BOD for a long period of time.

In this BOD product, the BOD and the stabilizer may be dissolved by adding a solvent to a container containing the BOD and the stabilizer to prepare a BOD solution. Therefore, the container in the present BOD product is preferably a container having resistance to the solvent.

(3-2) Example of Specific Embodiment of Present BOD Product The embodiment of the present BOD product is not particularly limited, but for example, the container has a bottle shape having an openable/closable lid, and the packaging is provided inside the lid portion. It is possible that the BOD and the stabilizer are stored inside the container (aspect A). In the case of aspect A, the stored BOD and stabilizer may be in liquid form or solid form. When the solid BOD and the stabilizer are stored in the embodiment A, the BOD and the stabilizer are added even after the solvent is added to the container to dissolve the BOD and the stabilizer. The BOD solution can be subsequently stably stored under a deoxygenated atmosphere by using the same container as that used for storage.

When the BOD and the stabilizer are solid such as powder, the packaged oxygen scavenger does not need to be provided on the lid, and may be enclosed together with the BOD and the stabilizer in the container. The oxygen scavenger in the container creates a deoxidizing atmosphere inside the container. Further, the air in the container may be replaced with an inert gas such as nitrogen.

Alternatively, the resin bag with the fastener may have a mode in which the BOD, the stabilizer, and the packaged oxygen absorber are stored.

(3-3) Application This BOD product can be used for any application. The present BOD products include, for example, hair dyeing, measurement of bilirubin concentration in biological samples, removal of bilirubin in biological samples, cosmetics, food processing, enzyme fuel cells (eg enzyme biobatteries, enzyme cells, etc.), and It can be used for crosslinking proteins. Since the stabilizer contained in the present BOD product does not inhibit the oxidation reaction by BOD, the present BOD product can be suitably used for the various uses described above, and can be preferably used for hair dyeing among the various uses.

[4. Hair dye]
Another embodiment of the present invention is described in [3. BOD product] is provided.

The BOD, the stabilizer, the solvent, the container, and the specific embodiment contained in the hair dye are described in the above [2. Storage method] and [3. The description in the section "BOD product" can be incorporated.

(4-1) Hair dyeability Since the stabilizer contained in the BOD product contained in this hair dye does not inhibit the oxidation reaction due to BOD, this hair dye has good hair dyeability. is there.

In the present specification, “good hair dyeability” means that the present BOD with respect to the color difference (ΔE* _ab ) of white hair after a hair dyeing test using a hair dye containing BOD and not containing a stabilizer. It means that the difference in color difference (ΔE* _ab ) of white hair after a hair dyeing test using a hair dye containing a product, that is, the reduction rate (%) of the color difference is 5.0% or less. That is, when the color difference (ΔE* _ab ) of the white hair after the hair dyeing test using the hair dye containing BOD and containing no stabilizer was 100%, the hair dye containing this BOD product was used. The relative value (%) of the color difference (ΔE* _ab ) of white hair after the hair dyeing test is 95.0% or more.

Here, the color difference (ΔE* _ab ) is a difference in hue between untreated gray hair and gray hair after the hair dyeing test, which is expressed using the L*a*b* color system. Here, L* represents lightness. There is a numerical value from 0 to 100, and the higher the numerical value, the brighter the image. In addition, a* indicates color. There is a numerical value of -60 to +60, with a negative sign indicating green and a positive sign indicating red. Further, b* indicates color. There is a numerical value of -60 to +60, with a negative sign indicating blue and a positive sign indicating yellow. The color difference, ΔE* _ab, is a value calculated by [(ΔL) ²⁺ (Δa) ² +(Δb) ² ] ^1/2 and is a unit generally used in the evaluation of hair color. The hair dyeing test will be described in the test example described later.

In one embodiment of the present invention, the reduction rate (%) of the color difference is preferably 5.0% or less, more preferably 4.0% or less, and further preferably 3.0% or less. It is preferably 2.0% or less, and particularly preferably 2.0% or less.

An embodiment of the present invention may have the following configuration.

[1] A method for storing bilirubin oxidase, which comprises storing a bilirubin oxidase solution containing bilirubin oxidase and a stabilizing agent in a deoxygenated atmosphere, wherein the stabilizing agent is the ABTS initial absorbance ( _A436 ) The reduction rate (%) is 0.10% or more, and the DMBQ initial absorbance (A ₂₅₆ ) reduction rate (%) is 0.80% or less.

[2] The container contains a bilirubin oxidase and a stabilizer, the inside of the container is a deoxygenated atmosphere, and the stabilizer has an ABTS initial absorption (A ₄₃₆ ) reduction rate (%) of 0.10% or more. And the reduction rate (%) of DMBQ initial absorbance (A ₂₅₆ ) is 0.80% or less, a bilirubin oxidase product.

[3] The bilirubin oxidase product according to [2], wherein the bilirubin oxidase and the stabilizer are in a solution state.

[4] A hair dye comprising the bilirubin oxidase product according to [2] or [3] above.

Hereinafter, one embodiment of the present invention will be specifically described by way of examples, but the present invention is not limited to these examples (test examples).

The present stabilizer and other substances (hereinafter referred to as comparative substances) used in this example are as follows. Only glyoxylic acid was adjusted to pH 7.0 with a potassium hydroxide solution and used in this example.

(Stabilizer)
・Uric acid (Wako Pure Chemical Industries)
・Glyoxylic acid (Wako Pure Chemical Industries)
・Powdered malt extract (manufactured by Kyokuto Pharmaceutical Co., Ltd.)
・Powdered fish extract (manufactured by Kyokuto Pharmaceutical Co., Ltd.)
・Powdered yeast extract (manufactured by Kyokuto Pharmaceutical Co., Ltd.)
CSL Sollis 095E (manufactured by Oriental Yeast Co., Ltd.) (referred to as CSL in Tables 1 to 4 and FIGS. 1, 2 and 4)
・Meast P1G (manufactured by Asahi Group Foods)
・Peptone A (manufactured by Kyokuto Pharmaceutical Co., Ltd.)
・Soybean flour 88 (Showa Sangyo)
・Yeast Extract (manufactured by Japan BD)
・Patty Case (Kyokuto Pharmaceutical Co., Ltd.)
・Hypolypeptone (Nippon Pharmaceutical Co., Ltd.)
・Peptone (Kyokuto Pharmaceutical Co., Ltd.)
(Comparative substance)
・Urea (Wako Pure Chemical Industries)
・Ammonium sulfate (manufactured by Nacalai Tesque)
・Dithiothreitol (DTT) (manufactured by Wako Pure Chemical Industries, Ltd.)
・Ammonium thioglycolate (made by Sasaki Chemical Co., Ltd.)
・Cysteamine hydrochloride (made by Sasaki Chemical Co., Ltd.)
Glyceryl monothioglycolate (described as GMT in Tables 1 to 3 and FIGS. 1 to 3) (manufactured by Sasaki Chemical Co., Ltd.)
・Sodium sulfite (manufactured by Wako Pure Chemical Industries, Ltd.)
・Glutathione (reduction type) (manufactured by Wako Pure Chemical Industries, Ltd.)
・L-Cysteine-hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.)
・N-acetyl-L-cysteine (manufactured by Wako Pure Chemical Industries, Ltd.)
-L-ascorbic acid (manufactured by Wako Pure Chemical Industries, Ltd.).

(Test Example 1) Storage of BOD by the present storage method A BOD solution containing BOD and the present stabilizer or comparative substance was kept in a non-deoxygenated atmosphere at 5° C. or in a deoxidized atmosphere for 30 days. Stored at °C. The stable storage of BOD according to the present storage method was evaluated by measuring the residual enzyme activity of BOD at any time during storage.

In Test Example 1, as a BOD, a BOD gene derived from Milocesium bergaria MT-1 (GenBank accession number: D125799.1) was introduced into the genus Aspergillus, and then a recombinant BOD expressed in the genus Aspergillus (hereinafter, referred to as (Also referred to simply as BOD) was used after purification using known techniques. The BOD gene derived from Milocesium bergaria MT-1 was introduced into the genus Aspergillus after codon optimization according to the gene sequence of the genus Aspergillus. Further, the purified BOD was dissolved in 50 mM glycine-KOH (pH 8.5) buffer so that the concentration of the BOD was 1.0 mg/mL, and used as a BOD enzyme solution.

The enzyme activity of BOD was measured as follows. A mixed solution (1980 μL) was prepared by adding 200 μL of 20 mM ABTS substrate solution to 1780 μL of 50 mM potassium phosphate buffer (pH 6.5), and the mixed solution was incubated at 37° C. for 1 minute. Thereafter, 20 μL of a solution containing BOD (specifically, a BOD storage solution for evaluation described later) was added to the mixed solution, and the resulting mixed solution (2 mL) was incubated at 37°C. Along with the incubation of the mixed solution, the absorbance at a wavelength of 436 nm of the mixed solution was measured every 20 seconds including immediately after the addition of the BOD storage solution for evaluation (0 second). From the absorbance at the wavelength of 436 nm of the mixed solution immediately after the addition of the evaluation BOD storage solution and the absorbance at the wavelength of 436 nm of the mixed solution after 1 minute from the addition of the evaluation BOD storage solution, 1 minute after the addition of the evaluation BOD storage solution The rate of increase in absorbance of the mixed solution at a wavelength of 436 nm was calculated. From the rate of increase, the BOD activity (unit) in the evaluation BOD storage solution was calculated. In the present specification, the amount of enzyme that oxidizes 1 μmol of ABTS per minute is defined as 1 unit.

The specific test method of Test Example 1 is as follows. One of the above-mentioned stabilizers was added to a BOD enzyme solution containing BOD to prepare a BOD storage solution (which is a BOD solution according to one embodiment of the present invention). Further, one kind of the above-mentioned comparative substance was added to the BOD enzyme solution to prepare a BOD stock solution. Furthermore, a BOD storage solution was prepared by adding distilled water to the BOD enzyme solution in place of the stabilizer or the comparative substance, and used as a control (not added in Table 1 and FIG. 1). The amount of the stabilizer or the comparative substance in the BOD storage solution is (a) when the molecular weight of the stabilizer or the comparative substance is clear such that the stabilizer or the comparative substance consists of a single compound, When the amount of the stabilizer or the comparative substance is 40 mM in the BOD stock solution, and (b) the stabilizer or the comparative substance is a substance composed of a composition whose molecular weight is unknown, 100 mL of the BOD stock solution, The amount of nitrogen element derived from the stabilizer or the comparative substance was 0.44 g. The amount of nitrogen element derived from the stabilizer or the comparative substance in the BOD storage solution was a value calculated by the Kjeldahl method. The final concentration of the stabilizer or comparative substance in each BOD stock solution is (a) “mM” for the stabilizer or comparative substance whose molecular weight is clear, and (b) the stabilizer or comparative substance whose molecular weight is unknown. The substances are shown in Table 1 as "percentage (%)" (amount (g) of stabilizer or comparative substance relative to 100 mL of BOD stock solution).

After that, each of these BOD storage solutions and an oxygen scavenger (Aneropack manufactured by Mitsubishi Gas Chemical Co., Inc.) were sealed in one container, and then the container was sealed to produce a BOD storage container. Storage of the BOD storage container was started in a non-deoxidized atmosphere and 5° C. or in a deoxidized atmosphere and 30° C. During the storage period, (a) a BOD storage solution was collected from the same BOD storage container at 0, 14, 21 and 35 days after the start of storage, or (b) at 0, 7, 18 and 33 days after the start of storage. Then, it was used as a BOD storage solution for evaluation. Here, after opening the BOD storage container for collecting the BOD storage solution for evaluation, when the BOD storage container was closed again, a new oxygen scavenger was used instead of the oxygen scavenger used until then. Enclosed with stock solution.

The BOD activity (unit) in the evaluation BOD storage solution was measured by the method described above. Regarding the activity of BOD, the relative value of the activity (unit) of BOD after the lapse of a predetermined number of days from the start of storage is defined as the residual activity (when the activity (unit) of BOD at the start of storage (day 0) is 100%). %). Regarding the residual activity, relative values (%) in other evaluation BOD storage solutions when the residual activity in the control evaluation BOD storage solution was 100% are shown in (a) and (b) of FIG. 1. .. The BOD activity (100%) at the start of storage (day 0) and the residual activity (%) of BOD 14 days after the start of storage are shown in (c) of FIG.

FIG. 1(a) shows that the residual activity of BOD was controlled when the BOD was stored by a storage method outside the scope of the present invention, that is, when the BOD solution was stored at 5° C. in a non-deoxygenated atmosphere ( It is a graph shown as a relative value (%) with respect to 100%. FIG. 1B shows a case where BOD is stored by a storage method according to an embodiment of the present invention or a storage method outside the scope of the present invention, that is, the BOD solution is stored at 30° C. in a deoxygenated atmosphere. It is a graph which shows the residual activity of BOD in the case of doing as a relative value (%) with respect to control (100%). FIG. 1( c) is a table showing the residual activity of BOD when BOD is stored using the storage method according to one embodiment of the present invention or a storage method outside the scope of the present invention. The residual activity of BOD is shown at time (day 0) and after 14 days from the start of storage.

From (a) and (b) of FIG. 1, when the BOD solution containing the present stabilizer was stored in a deoxygenated atmosphere, the BOD in the BOD solution was higher than that in the control (no addition). It was found to have higher enzyme activity (residual activity). That is, it was found that the present storage method can stably store BOD. More surprisingly, as shown in FIG. 1(c), it was found that according to the present preservation method, the activity of BOD was improved more than before preservation. On the other hand, it was found that the BOD solution containing urea, ammonium sulfate, DTT or GMT exhibits almost the same residual activity as the BOD solution without addition, even when stored in a deoxygenated atmosphere. That is, it was found that these urea, ammonium sulfate, DTT and GMT cannot stably store BOD.

In Test Example 1, (a) a BOD enzyme solution containing (a-1) BOD, and (a-2) a BOD stock solution containing one of the stabilizers, and (b) an oxygen scavenger It can be said that the BOD storage container obtained by enclosing the container in one container and then sealing the container is a BOD product in one embodiment of the present invention. That is, Test Example 1 is also the implementation of this stabilization method and can be said to be the production of this BOD product.

(Test Example 2) Evaluation of ABTS initial absorption ( _A436 ) reduction rate of stabilizer ABTS initial absorption ( _A436 ) reduction rate (%) of the stabilizer and comparative substance used in Test Example 1 was evaluated. ..

The specific evaluation method is as follows. ABTS manufactured by Roche Diagnostics was used as the ABTS. ABTS was dissolved in 50 mM glycine-KOH buffer (pH 8.5) so that the concentration of ABTS was 20 mM to prepare an ABTS solution. The absorbance of the ABTS solution at a wavelength of 436 nm (ABTS initial absorbance (A ₄₃₆ )) was measured with a molecular spectrophotometer SpectraMax Plus 384 (manufactured by Japan Molecular Device Co.) at an optical path length of 1.0 cm, and the absorbance was about 1. It was 3. Next, 5 μL of the stabilizer solution or the comparative substance solution was added to 195 μL of the ABTS solution to prepare a reaction solution for ABTS evaluation (hereinafter, also referred to as “reaction solution A”). Here, regarding the amount of each stabilizer or comparative substance in the reaction solution A, (a) the molecular weight of the stabilizer or comparative substance is clear so that the stabilizer or comparative substance consists of a single compound. In the case of the reaction solution A, the amount of the stabilizer or the comparative substance is 1 mM, and when (b) the stabilizer or the comparative substance is a substance composed of a composition whose molecular weight is unknown, 100 ml of the reaction solution A is added. The amount of nitrogen element derived from the stabilizer or the comparative substance was 0.011 g. The final concentration of the stabilizer or the comparative substance in the reaction solution A is (m) for the stabilizer or the comparative substance whose molecular weight is clear, and (b) the stabilizer or the comparative substance whose molecular weight is unknown. "Percentage (%)" (amount (g) of stabilizer or comparative substance relative to 100 mL of reaction solution A) is shown in Table 2.

Then, immediately after preparation of the reaction solution A, that is, immediately after addition of the stabilizer solution or the comparative substance solution to the ABTS solution, the reaction solution A was reacted at 25° C. for 1 minute. Immediately thereafter, the absorbance of the reaction solution A at a wavelength of 436 nm was measured using a molecular absorptiometer of the same type as that used for measuring the absorbance of the ABTS solution. Further, instead of the stabilizer solution or the comparative substance solution, 5 μL of distilled water was added to 195 μL of the ABTS solution to prepare a reaction solution A containing no stabilizer or the comparative substance, and a control solution (Table 2 and FIG. 2) was prepared. Then, it is described as no addition). Also for the control, after the reaction solution A was reacted at 25° C. for 1 minute, the absorbance of the reaction solution A at a wavelength of 436 nm was measured using the above-mentioned molecular absorptiometer. The initial absorbance of ABTS (A ₄₃₆ ) thus obtained was compared with the absorbance of the reaction solution A at a wavelength of 436 nm. Absorbance decrease rate (%) per minute after addition of the stabilizer or comparative substance of reaction solution A to ABTS initial absorbance (A ₄₃₆ ) (100%) was calculated, and ABTS initial absorbance (A ₄₃₆ ) decrease rate (%). The results are shown in Table 2 and FIG.

Table 2 shows the absorbance at a wavelength of 436 nm of the reaction solution A after the addition of the stabilizer or the comparative substance according to one embodiment of the present invention with respect to the absorbance at a wavelength of 436 nm of the ABTS solution (ABTS initial absorbance (A ₄₃₆ )). shows a reduction rate per minute (ABTS initial absorbance _{(a 436)} reduction rate (%)). FIG. 2 is a diagram showing a reduction rate (%) of ABTS initial absorption (A ₄₃₆ ) by a stabilizer or a comparative substance according to an embodiment of the present invention. Specifically, the results of Table 2 are shown as a graph. Shows.

In FIG. 2, it was found that the initial absorbance of ABTS did not change in the reaction solution A containing the comparative substance or the reaction solution A containing no stabilizer. On the other hand, it was found that the reaction solution A containing the present stabilizer had an ABTS initial absorbance decrease rate of 0.10% or more.

(Test Example 3) Evaluation of DMBQ initial absorbance (A ₂₅₆ ) reduction rate of stabilizer The DMBQ initial absorbance (A ₂₅₆ ) reduction rate (%) of the stabilizer and comparative substance used in Test Example 1 was evaluated. ..

The comparative substances used in Test Example 3 are substances other than the stabilizer described in Table 3 and FIG. 3 described later. The stabilizers used in Test Example 3 were uric acid, powdered malt extract, powdered fish extract, powdered yeast extract, CSL, mist P1G, peptone A, soybean powder 88, Yeast Extract, Patticase, Hippolypeptone and peptone. In Table 3 and FIG. 3, since the results of the stabilizers used in Test Example 3 were the same, they were collectively referred to as “stabilizer”.

The specific evaluation method is as follows. As the DMBQ, DMBQ manufactured by Tokyo Chemical Industry Co., Ltd. was used. First, a 1 mM DMBQ solution was prepared. The absorbance (A ₂₅₆ ) of a 1 mM DMBQ solution at a wavelength of 256 nm was measured with an ultraviolet-visible spectrophotometer UV-160A (manufactured by Shimadzu Corporation) at an optical path length of 1.0 cm, and the absorbance was about 1.8. Met. Next, to 890 μL of distilled water, (a) 1 mM DMBQ solution (100 μL) and (b) stabilizer solution or comparative substance solution (10 μL) were added, and a reaction solution for DMBQ evaluation (hereinafter, also referred to as reaction solution D). ) Was produced. Here, the amount of each stabilizer or comparative substance in the reaction solution D is (a) the molecular weight of the stabilizer or comparative substance is clear so that the stabilizer or comparative substance consists of a single compound. In the case of the reaction solution D, the amount of the stabilizer or the comparative substance is 0.1 mM, and (b) when the stabilizer or the comparative substance is a substance having a composition of unknown molecular weight, the reaction solution The amount of the nitrogen element derived from the stabilizer or the comparative substance was 0.0011 g in 100 mL of D. The final concentration of the stabilizer or comparative substance in the reaction solution D is (m) for the stabilizer or comparative substance whose molecular weight is clear, and (b) the stabilizer or comparative substance whose molecular weight is unknown. “Percentage (%)” (amount (g) of stabilizer or comparative substance relative to 100 mL of reaction solution D) is shown in Table 3. Further, instead of the stabilizer solution or the comparative substance solution, a reaction solution D using 10 μL of distilled water was prepared and used as a control (not added in FIG. 3).

The absorbance of the reaction solution D at a wavelength of 256 nm was measured using (a) the reaction solution D immediately after the preparation (that is, immediately after the test was started, which is indicated as 0 minute in FIG. 3A), and (b) the reaction solution D of 25 nm. The measurement was performed every 10 seconds after standing at 0° C. using an ultraviolet-visible spectrophotometer UV-160A. Here, the absorbance at a wavelength of 256 nm of the reaction solution D immediately after preparation was defined as the DMBQ initial absorbance (A ₂₅₆ ). From the measurement results, the absorbance decrease rate (%) per minute after the addition of the stabilizer or the comparative substance of the reaction solution D (after the start of the test) to the DMBQ initial absorbance (A ₂₅₆ ) (100%) was calculated, DMBQ initial absorbance (A ₂₅₆ ) reduction rate (%) was used. The results are shown in Fig. 3.

FIG. 3 is a diagram showing a DMBQ initial absorbance (A ₂₅₆ ) reduction rate (%) by a stabilizer or a comparative substance according to an embodiment of the present invention. Specifically, FIG. 3A shows the stabilization according to the embodiment of the present invention when the absorbance of the reaction solution D at the wavelength of 256 nm is 100% immediately after the start of the test (0 minutes). It is a figure which shows the relative reduction rate (%) of the light absorbency after the addition of an agent or a comparative substance (after the start of a test). FIG. 3B shows the absorbance of the reaction solution D at a wavelength of 256 nm after the addition of the stabilizer or the comparative substance according to one embodiment of the present invention with respect to the absorbance immediately after the start of the test (0 minutes) (after the start of the test). FIG. 3 is a diagram showing the absorbance decrease rate (%) per minute () (DMBQ initial absorbance (A ₂₅₆ ) decrease rate (%)) of FIG.

For each of the stabilizers used in Test Example 3, (1) the relative decrease rate (%) of the absorbance of the reaction solution D at a wavelength of 256 nm (FIG. 3A) was the same, and the results were 100%. The results of (2) DMBQ initial absorbance (A ₂₅₆ ) reduction rate (%) (Fig. 3(b)) were all the same and were below the detection limit (0.00%).

(Test Example 4)
When the hair dye according to an embodiment of the present invention is used for hair dyeing, a hair dyeing test using human white hair was performed for the effect of the stabilizer contained in the hair dye on the hair dyeing property. Evaluated. As a control test, the same evaluation was performed using a hair dye containing L-ascorbic acid and BOD instead of the hair dye according to one embodiment of the present invention.

(Preparation of hair dye)
POE (20) hydrogenated castor oil, lactic acid, and para-phenylenediamine (hereinafter, also referred to as PPD) as an oxidative dye were each dissolved in a predetermined amount of distilled water, and then mixed to prepare a mixed solution. Then, the pH of the mixed solution was adjusted to pH 8.5 using monoethanolamine. Next, a hair dye was prepared by adding (a) L-ascorbic acid or a stabilizer, (b) BOD enzyme solution, and (c) distilled water to the above mixed solution. In addition, a hair dye was prepared without adding L-ascorbic acid or a stabilizer, and used as a control (not added in FIG. 4). “POE” is an abbreviation for “polyethylene oxide”.

Here, L-ascorbic acid was added to the mixed solution so that the concentration of L-ascorbic acid in the hair dye would be 8 mM. As the stabilizer, uric acid, powdered fish extract, CSL, mist P1G, Yeast Extract, Hippolypepton and powdered malt extract were used. The stabilizer was added to the mixed solution so that the concentration of the stabilizer in the hair dye (the amount (g) of the stabilizer in 100 g of the hair dye) was the concentration shown in Table 4. Further, POE(20) hydrogenated castor oil, lactic acid and PPD had a concentration of POE(20) hydrogenated castor oil, lactic acid and PPD in the hair dye of 2.0% each relative to 100% by weight of the hair dye. %, 1.0 wt%, and 0.3 wt% were mixed and used.

(Hair dyeing test)
For each bundle of human white hair (10 cm, 1 g, manufactured by Vyrax) (hereinafter, also referred to as hair), 2 g of the hair dye prepared as described above was applied to the hair. After application, the hair was left at 30° C. for 10 minutes, then the hair was turned inside out and left for another 10 minutes. Then, the hair was washed with running water, washed with 1% SDS, and then washed with running water to remove 1% SDS of the hair. The water on the washed hair was wiped off with a towel, and the hair was air-dried overnight.

(Hue evaluation)
Using the color-difference meter CR-400 (manufactured by Konica Minolta Co., Ltd.), the hue of the white hair that has not been subjected to the hair dyeing test (untreated white hair) and the white hair after the hair dyeing test described above is C It measured using the light source. Based on the measurement results, the color difference (ΔE* _ab ) between the untreated white hair and the white hair after the hair dyeing test was calculated using the L*a*b* color system. The results are shown in Fig. 4 and Table 4.

Table 4 shows the case where a hair dye containing L-ascorbic acid or the present stabilizer is used, where the color difference (ΔE* _ab ) when using an additive-free hair dye is 100(%). The relative value (%) of the color difference (ΔE* _ab ) is shown. FIG. 4 shows the results of a hair dye test when a hair dye containing L-ascorbic acid or a hair dye containing the stabilizer (ie, the hair dye) was used. Specifically, FIG. 4A is a photograph of a sample (white hair) after hair dyeing with various hair dyes. Untreated is white hair that has not been subjected to a hair dye test. FIG. 4B shows the result (color difference (ΔE* _ab )) of the hue evaluation of the white hair after dyeing.

The following facts were found from FIG. 4 and Table 4. Relative value of color difference (ΔE* _ab ) when using a hair dye containing this stabilizer, where the color difference (ΔE* _ab ) when using an additive-free hair dye is 100(%) (%) was 95.0% or more. That is, the hair dye containing the present stabilizer hardly inhibited the hair dyeing property. On the other hand, in the case where the color difference in the case of using the hair dye of no addition of (Delta] E * _ab) 100 and (%), the color difference in the case of using a hair dye containing L- ascorbic acid (Delta] E * _ab) The relative value (%) was 76.0%. That is, it was found that L-ascorbic acid had a high inhibitory effect on hair dyeing.

From the above results, according to the present storage method, BOD can be stably stored by storing the BOD solution containing the stabilizer according to one embodiment of the present invention together with BOD in a deoxygenated atmosphere. , Was shown. In addition, when the present stabilizer is used for a hair dye, in other words, when the present hair dye containing the present BOD product is used, compared to the case where no additive hair dye is used. The reduction rate of the color difference of the white hair after the test was 5% or less. Therefore, it was shown that the present stabilizer contained in the present hair dye does not affect hair dyeing.

According to one embodiment of the present invention, BOD can be stably stored without inhibiting the oxidation reaction by BOD. Therefore, the present invention is applicable to a wide range of fields using BOD, such as the field of hair dyeing, the field of clinical examination, the field of cosmetics, the field of enzyme fuel cells (eg, enzyme biobatteries, enzyme cells, etc.), and the field of food processing. It can be used suitably.

Claims

A method for storing bilirubin oxidase, which comprises storing a bilirubin oxidase solution containing bilirubin oxidase and a stabilizer in a deoxygenated atmosphere:
Here, the above stabilizer has an ABTS initial absorption ( A436 ) reduction rate (%) of 0.10% or more, and a DMBQ initial absorption ( A256 ) reduction rate (%) of 0.80% or less. Is.
Contains bilirubin oxidase and a stabilizer in the container,
The inside of the container is a deoxygenated atmosphere,
The above stabilizer has an ABTS initial absorption ( A436 ) reduction rate (%) of 0.10% or more, and a DMBQ initial absorption ( A256 ) reduction rate (%) of 0.80% or less. A bilirubin oxidase product characterized by:
The bilirubin oxidase product according to claim 2, wherein the bilirubin oxidase and the stabilizer are in a solution state.
A hair dye comprising the bilirubin oxidase product according to claim 2 or 3.