JP2007107148A - Method for utilizing peroxidase derived from rice - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a peroxidase having high reactivity and stability at a wide hydrogen peroxide concentration, to provide a method for carrying out wastewater treatment in the textile industry, etc., accompanied with dye migration inhibition in a washing or a rinsing process and bleaching of a fiber dye in a solution or a dispersion and to provide a composition used for the method. <P>SOLUTION: The peroxidase having the high reactivity and stability at the wide hydrogen peroxide concentration is discovered to confirm its effects. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for decomposing (decoloring / bleaching) colored substances (dyes, etc.) in a solution using a peroxidase exhibiting stable reactivity and storage stability in the presence of hydrogen peroxide, and a composition.

Peroxidase (EC 1.11.11.7) is an enzyme that acts as an oxidation catalyst for a substrate (electron or hydrogen donor) using hydrogen peroxide as an oxidizing agent. Although peroxidase is classified into several types depending on the prosthetic group and the reaction format, the use of heme-type peroxidase containing heme as the prosthetic group is best known in the industry.
For industrial use of peroxidase, polymer synthesis by polymerization reaction, synthesis of oxidized compounds by oxidation reaction, decomposition of various organic compounds, for example, use in the manufacturing process of paper pulp, bleaching and decomposition of fiber dyes A wide range of proposals have already been made. In these known methods, peroxidase is usually used together with hydrogen peroxide or a compound or enzyme system capable of generating hydrogen peroxide in situ. Hydrogen peroxide is an oxidizing agent for peroxidase, and is necessary for forming reactive species, and at the same time, has the property of oxidizing not only peroxidase but all enzymes and losing its function. Therefore, a peroxidase having stable reactivity and storage stability with respect to a wide range of concentrations of hydrogen peroxide has been strongly desired for industrial use, but has not yet been achieved. The same applies to the case of fiber dye bleaching / degradation as shown below.

  Peroxidases have been proposed for use as dye transfer inhibitors and fiber dye bleaches. Here, “dye transfer prevention” is a process comprising, for example, prevention of migration of fiber dye from a dyed cloth to another cloth as described in Japanese Patent Publication No. 2801398. This dye transfer problem is important when washing together dark and white or light fabrics where the dye oozes during washing. Furthermore, based on the bleaching action of peroxidase on fiber dyes in solution or dispersion, the enzyme is expected to be effective in wastewater treatment from the textile industry. It is also known that these capabilities of peroxidase are particularly remarkable when combined with hydrogen peroxide and an oxidizable substrate other than a dye called an activator.

  As peroxidases that actually have an effect on dye transfer inhibition and bleaching, peroxidases derived from plants such as horseradish, soybean, rice (see Patent Document 1) or basidiomycete Colprinus cinereus have already been proposed.

  Unfortunately, the peroxidase activity level in the natural environment is often not maintained in a wash or wastewater treatment environment. In particular, the various properties of peroxidases such as reactivity (reaction rate), thermal stability, pH stability, oxidation stability and substrate specificity are relatively unnatural conditions in washing and wastewater treatment environments. It is not always optimal for use or storage as a detergent composition such as a granulated product, and for storage after processing.

  For example, peroxidase derived from Colprinus cinereus has a property that its reaction rate is faster than known peroxidases. Furthermore, it does not act on dyes in clothing, but it works well on pigments released in washing water and has the ability to decolorize. Therefore, as an industrial application, it has been considered to prevent color migration during washing. It was. Although it has such excellent characteristics, it is practically used because it is rapidly deactivated at 50 ° C. at high pH (10.5), high concentration of hydrogen peroxide (5-10 mM), which is one of the conditions during washing. It is known that problems to be solved remain (see Patent Document 2 and Non-Patent Document 2).

  Due to advances in genetic engineering today, it is possible and relatively easy to substitute a specific amino acid residue in an amino acid sequence of a protein with another amino acid residue. Depending on the position of the amino acid residue to be substituted, the chemical nature and physical size of the substituted amino acid residue, etc., the performance of the original enzyme can be changed to various degrees, and in some cases Or even inactivate the original enzyme.

  Intensive research has been conducted to solve the above problem by preparing a mutant enzyme in which mutations are randomly introduced in the peroxidase derived from Colprinus cinereus. Researchers created mutant enzyme populations, and selected those with high residual activity even after inactivation treatment simulating washing conditions of pH 10.5, 15 mM hydrogen peroxide, 50 ° C., 20 minutes. Furthermore, a novel mutant enzyme group was selected by further mutating the obtained mutant enzyme or combining effective mutations. As a result, the wild-type enzyme had a residual activity of 0% after inactivation treatment at pH 10.5, 15 mM hydrogen peroxide, 50 ° C. and 20 minutes, whereas the residual activity after inactivation treatment was 39%. A group of mutant enzymes represented by a novel mutant enzyme has been created. Although it has been shown that the dye transfer blocking effect has been enhanced by the contribution of the improvement in the residual activity, unfortunately, the practical activity level has not yet been reached due to insufficient residual activity. In other words, it can be used as a dye transfer inhibitor and a fiber dye bleaching agent having a high residual activity at a pH of 10.5, a hydrogen peroxide concentration of 15 mM, and a high temperature (50 ° C.), which is one of washing conditions. The presence of peroxidase is not known.

  Another solution to this is to design and substantially improve stability against hydrogen peroxide attack. One way to achieve this goal is described in international patent application WO 94/18314, in which an oxidation sensitive amino acid (eg methionine, tryptophan, cysteine or tyrosine) is removed from the amino acid sequence of the enzyme. Or other amino acids with higher oxidative stability are introduced instead of those amino acids. A similar method is described in international patent application WO 95/21247, which recommends replacing at least one methionine in the amino acid sequence of the enzyme with an amino acid that is neither methionine nor cysteine. ing.

  In any case, an enzyme that is stable under extremely unnatural conditions for enzymes such as high pH (10.5), high concentration hydrogen peroxide (15 mM), and high temperature (50 ° C.), which is one of the conditions at the time of washing. Although strongly desired, it has not yet been realized even if genetic engineering techniques are used, and the existence of a wild-type enzyme having such characteristics has not been expected.

  In addition, in order to enhance the washing effect at the time of washing, a method of leaving it in a washing solution for several minutes to several hours in advance is widely known as post-washing, but peroxidase that can be expected to work stably in that environment The existence of is not known.

  Furthermore, a cleaning method that improves the cleaning power against sebum dirt and circulates the yellowing of clothing by circulating a cleaning solution with a high detergent concentration through clothing is becoming widespread, but it can be expected to work stably in that environment. The existence of is also unknown.

  In addition, the presence of peroxidase that can be expected to exhibit excellent stability against hydrogen peroxide during processing as a detergent composition such as granulation and during storage after processing has not been known.

WO2004 / 033610 US Pat. No. 5,968,883 The 35th Symposium on Cleaning (Japan Oil Chemists' Society) Tokyo, pages 105-106 Nature Biotechnology 1999, 17, p379-384

  In view of the above-mentioned background art, the object of the present invention is included when used for wastewater treatment in the textile industry, etc., which involves dye transfer prevention in washing or rinsing processes and bleaching of dyes in solutions or dispersions. (2) A peroxidase having a sufficient reaction rate and (2) a low rate of decrease in the reaction rate within the expected reaction time is found and used. Is to provide a way.

  Another object of the present invention is to find a peroxidase exhibiting excellent stability against hydrogen peroxide at the time of processing as a detergent composition such as granulation and storage after processing, and to provide a method of using this.

  The present inventors surprisingly show that rice-derived chaff peroxidase has a residual activity of 55% even after inactivation treatment at pH 10.5, high temperature (50 ° C.), 15 mM hydrogen peroxide, 20 minutes. I found out. Further, the present inventors have found that even if the hydrogen peroxide concentration is an extremely high concentration of 375 mM, it is possible to maintain an effective activity level for dye migration inhibition and bleaching over 20 minutes or more. Furthermore, it has been found that even if the hydrogen peroxide concentration is as high as 1M, it has a function as a catalyst. In addition, it was confirmed that the detergent composition was stable even when stored for 4 weeks with a hydrogen peroxide source.

The present invention has been completed based on the above findings. That is, the present invention is as follows.
(1) A method for preventing migration of fiber dye from one dyed cloth to another cloth in the case of washing and / or rinsing together a plurality of cloths including cloth dyed in washing liquid And a method of allowing a rice peroxidase having hydrogen peroxide resistance to act on the washing solution in the presence of hydrogen peroxide.
(2) A method of bleaching a fiber dye in a solution or dispersion, wherein a rice-derived peroxidase having hydrogen peroxide resistance is allowed to act on the solution or dispersion in the presence of hydrogen peroxide. .
(3) The method according to any one of claims 1 and 2, wherein the concentration of hydrogen peroxide is 0.001 mM or more and 1 M or less.
(4) A method characterized by oxidizing rice or a hydrogen donor by causing a peroxidase derived from rice to act in a hydrogen peroxide concentration range of 0.001 mM to 1 M.
(5) The method according to any one of (1), (2), (3) and (4), wherein the rice-derived peroxidase is a rice bran-derived peroxidase.
(6) Hydrogen peroxide acts as a hydrogen peroxide precursor, a coexistence system of a bleach activator and a hydrogen peroxide precursor that can generate hydrogen peroxide by acting on the hydrogen peroxide precursor, and generate hydrogen peroxide. 6. The method according to any one of claims 1, 2, 3, 4, or 5, wherein the enzyme system is supplied by at least one of the possible enzyme systems. The method described.
(7) A detergent composition or a bleaching composition containing rice-derived peroxidase having hydrogen peroxide resistance.
(8) The composition according to claim 7, wherein the rice-derived peroxidase is a rice bran-derived peroxidase.
(9) The composition according to claim 7 or 8, wherein the concentration of hydrogen peroxide is 0.001 mM or more and 1 M or less when used as a detergent or a bleaching agent.
(10) Hydrogen peroxide generates a hydrogen peroxide precursor, a coexistence system of a bleach activator that can act on the hydrogen peroxide precursor and generate hydrogen peroxide, and a hydrogen peroxide precursor, and hydrogen peroxide. 10. A composition according to any one of claims 7, 8, or 9, characterized in that it is supplied by means of at least one of the possible enzyme systems.

  Reactions in a wide range of hydrogen peroxide concentrations can be expected by using perioxidase derived from rice bran that shows high reactivity and stability to hydrogen peroxide up to 1M. Rice-derived peroxidase has another preferable property of being excellent in heat resistance (described in WO 2004/033610), so that it can provide a wide range of applications particularly in fiber dye migration prevention and bleaching. In addition, it becomes possible to provide a composition containing peroxidase exhibiting excellent stability during processing as a detergent composition such as granulation and during storage during processing.

  The present invention relates to a method for preventing the transfer of fiber dye from a dyed fabric to another fabric in the case of washing and / or rinsing the fabric together in a washing solution, wherein the fabric is washed and / or rinsed. A peroxidase derived from rice having hydrogen peroxide resistance, and a bleach activator capable of generating hydrogen peroxide by acting on the hydrogen peroxide, a hydrogen peroxide precursor, or a hydrogen peroxide precursor. The present invention relates to a method characterized in that a coexisting system with a hydrogen peroxide precursor or an enzyme system capable of generating hydrogen peroxide is present substantially simultaneously.

  The present invention also relates to a method for bleaching a fiber dye in a solution or dispersion, wherein the solution or dispersion includes a hydrogen peroxide-resistant rice peroxidase, hydrogen peroxide, or hydrogen peroxide. A coexisting system of a precursor or a bleach activator capable of acting on a hydrogen peroxide precursor to generate hydrogen peroxide and a hydrogen peroxide precursor, or an enzyme system capable of generating hydrogen peroxide. It is related with the method characterized by making it exist simultaneously.

  Further, the present invention relates to a rice-derived peroxidase having hydrogen peroxide resistance, and bleach activation that can act on hydrogen peroxide, a hydrogen peroxide precursor, or a hydrogen peroxide precursor to generate hydrogen peroxide. The present invention relates to the provision of a detergent composition or a bleaching composition containing a coexisting system of an agent and a hydrogen peroxide precursor or an enzyme system capable of generating hydrogen peroxide substantially simultaneously.

  Although the amount and quality of impurities contained in the rice-derived peroxidase having hydrogen peroxide resistance in the present invention varies depending on the degree of purification, the RZ value is preferably 0.01 or more, more preferably 0. One or more, more preferably 1.0 or more may be used. The RZ value here is a ratio of absorbance at 275 nm and 403 nm (A275 nm / A403 nm). This value represents the ratio of the heme content to the protein content in the peroxidase solution and is an indicator of the degree of purification of the peroxidase.

  The rice-derived peroxidase having hydrogen peroxide resistance in the present invention can be exemplified by rice bran-derived peroxidase that is separated, purified, and recovered from rice straw containing the enzyme, but has hydrogen peroxide resistance. If it is a rice-derived peroxidase, there will be no restriction | limiting in particular regarding the localized part. For example, even rice-derived peroxidase derived from straw or foliage is regarded as a rice-derived peroxidase having hydrogen peroxide resistance as long as it has hydrogen peroxide resistance. In the present invention, if any one of the following conditions is satisfied, it is determined that the compound has hydrogen peroxide resistance. (Condition 1) 0.1 M Tris buffer pH 10.5, hydrogen peroxide concentration 15 mM, 50 ° C. After 20 minutes of inactivation treatment, hydrogen peroxide can be used as long as 50% or more of the pre-treatment activity is maintained. Judged to be resistant. (Condition 2) 0.1M Tris buffer pH 10.5, hydrogen peroxide concentration 15 mM, reaction rate constant at 0.1 M Tris buffer pH 10.5, hydrogen peroxide concentration 15 mM even after 20 minutes of inactivation treatment Is determined to be resistant to hydrogen peroxide. (Condition 3) If the residual activity of 1% or more is maintained even after inactivation treatment at pH 10.5, hydrogen peroxide concentration 375 mM, 50 ° C., and 20 minutes, it has hydrogen peroxide resistance. judge. The case where 50% or more of activity is retained as in the examples described later is the most preferable case. (Condition 4) If activity having a reaction rate constant of 0.2 or more is maintained at pH 10.5, hydrogen peroxide concentration 1M, and 50 ° C., it is determined that the compound has hydrogen peroxide resistance.

  Rice-derived peroxidase in the present invention, particularly rice bran-derived peroxidase, can be easily obtained from rice bran by the purification method described in Japanese Patent Publication No. 5-22510.

  Furthermore, due to recent results of rice genome analysis and the like, the presence of multiple peroxidase genes has also been reported in rice (Phytochemistry 65, pp.1879-1893 (2004)). That is, using today's genetic engineering techniques, a combination of a DNA sequence encoding these rice peroxidases and a DNA sequence having the function of expressing the peroxidase-encoding DNA sequence is combined. It is also possible to construct a replacement DNA molecule and then transform a host cell or host plant with the recombinant DNA molecule to efficiently produce rice peroxidase. In view of the technical background as described above, the transformed host cell is cultured, or the transformed host plant body is grown, and the obtained culture, cultured cell, or plant body is used. The rice-derived peroxidase having hydrogen peroxide resistance obtained by separating, purifying and recovering peroxidase is also included in the category of the rice-derived peroxidase having hydrogen peroxide resistance of the present invention.

  There is no particular limitation on the form of the rice-derived peroxidase having hydrogen peroxide resistance in the present invention, but considering the case where it is added as a component of a detergent composition or a bleaching composition, dust-free granules or liquid enzyme preparations It is preferable that it is a form. In the case of dust-free granules, they are made as described in US Pat. Nos. 4,106,991 and 4,661,452, and can be optionally coated by methods known in the art. . In the case of a liquid enzyme preparation, it can be stabilized by adding a polyol such as propylene glycol, sugar or sugar alcohol, lactic acid or boric acid or the like according to an established method.

  When washing and / or rinsing the cloth together in the washing solution of the present invention, or bleaching the fiber dye in the solution or dispersion, 0.001 mg to 1000 mg of rice per liter of washing solution or bleaching solution. Derived peroxidase may be added. Furthermore, the temperature may be in the range of 10 ° C to 80 ° C, preferably in the range of 20 ° C to 70 ° C, more preferably in the range of 20 ° C to 60 ° C. Furthermore, the pH of the washing solution or the bleaching solution is a general pH, a general pH in the case of washing and / or rinsing, that is, pH 6.5 to 10.5, preferably 6.5 to 9.5, more preferably 7 What is necessary is just to implement by 5-9.5.

  In the present invention, in order to prevent dye migration in the washing or rinsing process and to bleach the fiber dye in the solution or dispersion, it is essential that the rice peroxidase and hydrogen peroxide are present substantially simultaneously. is there. In this case, hydrogen peroxide may be present in an amount of 0.001 to 1.0 M at the beginning or during the washing and / or rinsing process. Preferably, it may be present at 25 mM to 1.0 M. Alternatively, it may be present at 110 mM to 1.0 M, more preferably 330 mM to 1.0 M, and even more preferably 400 mM to 1.0 M. Alternatively, it may be present at 600 mM to 1.0 M. Further, it may be present at 800 mM to 1.0 M.

  In supplying hydrogen peroxide, hydrogen peroxide itself may be added to the washing solution, solution or dispersion, or may be supplied as a hydrogen peroxide precursor that generates hydrogen peroxide in situ.

  Representative hydrogen peroxide precursors include perborate, percarbonate, peroxycarboxylic acid, or a salt thereof. Specific examples include sodium perborate and sodium percarbonate.

  Further, a bleaching activator capable of acting on the hydrogen peroxide precursor and generating hydrogen peroxide may coexist with the hydrogen peroxide precursor. Specific examples of such bleach activator include nonanoyloxybenzenesulfonate sodium salt, dodecanoyloxybenzenesulfonate sodium salt, 4-decanoyloxybenzoic acid, tetraacetylethylenediamine, and the like. . The amount of bleach activator to be present is suitably between about 1 μM and 10 mM, more preferably between about 10 μM and 1 mM, at the beginning or during the washing and / or rinsing process.

  Furthermore, a hydrogen peroxide generating enzyme system that generates hydrogen peroxide on site may be selected as a hydrogen peroxide supply source. A preferred hydrogen peroxide producing enzyme system is one that acts on an inexpensive and readily available substrate (hydrogen peroxide precursor) that is conveniently included in a detergent composition. An example of such a substrate (hydrogen peroxide precursor) is glucose, which can supply hydrogen peroxide by using glucose oxidase. Other suitable oxidases are urate oxidase, galactose oxidase, alcohol oxidase, amine oxidase, amino acid oxidase and cholesterol oxidase.

  In the present invention, the dye transfer inhibition and bleaching effect of the peroxidase derived from rice having hydrogen peroxide resistance is enhanced when the dye transfer inhibition in the washing or rinsing process and the fiber dye in the solution or dispersion are bleached. In order to achieve this, another oxidizing substrate may coexist. These oxidizing substrates are generally referred to as activators, and specific examples include p-iodophenol, L-ascorbic acid, p-aminophenol, p-aminobenzoic acid substrate, and the like. It is not a thing. The amount of the oxidizable substrate to be coexisted is about 1 μM to 10 mM, more preferably about 10 μM to 1 mM.

  In the present invention, in the case of performing dye transfer prevention in the washing or rinsing process and bleaching of the fiber dye in the solution or dispersion, an anionic surfactant, nonionic surfactant, cationic surfactant, amphoteric interface An activator and a mixture of these surfactants may coexist. Examples of such surfactants include alkylbenzene sulfonate (LAS: anionic surfactant), α-olefin sulfonate (AOS: anionic surfactant), and alkyl polyoxyethylene sulfate (AES: Anionic surfactant) Sodium dodecyl sulfate (SDS: anionic surfactant) Long chain fatty acid salt such as sodium laurate (soap: anionic surfactant), Brij35 (nonionic surfactant), alkylpolyoxy Examples include ethylene ether (nonionic surfactant), alkyltrimethylammonium salt (cationic surfactant), dialkyldimethylammonium salt (cationic surfactant), and dimethylalkylamine oxide (amphoteric surfactant). It is not limited to these. Further, the amount of the surfactant to be coexistent is not particularly limited, but is preferably not less than the critical micelle concentration (CMC) of each surfactant.

  In the present invention, there is no particular limitation on the fiber dye that is subject to dye transfer inhibition and bleaching by the rice-derived peroxidase having hydrogen peroxide resistance, and the fiber dye that is usually used, including in the coexistence with the aforementioned activator. Is effective against The fiber dye referred to here is not limited to natural dyes, but is synthetically produced, but has the same structure and properties as natural dyes, including common azo dyes, general azo dyes, and anthraquinones. Mention may be made of other synthetic dyes such as dyes. It is also effective for reactive dyes that are covalently bonded to fibers.

  Finally, the present invention relates to a composition comprising a rice-derived peroxidase that is resistant to hydrogen peroxide. Specifically, it relates to a detergent composition or a bleaching composition. In addition, coexistence with hydrogen peroxide or a component intended for its supply is essential. For this purpose, hydrogen peroxide precursor, bleach activator or hydrogen peroxide-producing enzyme system is one of the components. You may choose. That is, the present invention relates to a rice-derived peroxidase having hydrogen peroxide resistance, a hydrogen peroxide, a precursor of hydrogen peroxide, a bleach activator that acts on the hydrogen peroxide precursor to generate hydrogen peroxide and a peroxidase. The present invention relates to a detergent composition or a bleaching agent composition comprising at least one of a coexisting system with a hydrogen oxide precursor and an enzyme system capable of generating hydrogen peroxide. Of course, a surfactant for enhancing the cleaning effect may be included.

  There is no particular limitation on the amount of hydrogen-derived rice peroxidase in the composition, but preferably 0.001 mg to 1000 mg of rice-derived peroxidase is added per liter of washing solution, solution, or dispersion. Just do it.

  There is no particular limitation on the form, and either a powder form or a liquid form may be selected so as to be convenient for the purpose. Specifically, liquid enzyme preparations stabilized with dust-free granules as described above and various stabilizers can be selected. In particular, in the case of powder, a composition composed of a granulated product in which all the components in the composition are mixed, a granulated product of individual components in the composition, or a mixed granulated product of arbitrary plural components are mixed. It may be a composition. In addition, the mixed granulated product of arbitrary plural components mentioned here is, for example, a granulated product obtained by mixing rice-derived peroxidase and a bleach activator capable of generating hydrogen peroxide by acting on a hydrogen peroxide precursor. It is a grain.

  In addition, when the detergent composition is intended, other detergent components known in the art, such as builders, anticorrosives, sequestering agents, anti-fouling redeposition agents, fragrances Other detergent enzymes (eg proteases, lipases or amylases) such as enzyme stabilizers may be included.

  The pH of the washing liquid to which the detergent composition of the present invention is added or the bleaching liquid to which the bleach composition is added is 6.5 to 12, preferably 6.5 to 10.5.

  Next, the present invention will be described in more detail based on the following examples, but the present invention is not limited to the following examples.

[Example 1] Effect of 20 minutes of inactivation treatment at 50 ° C., pH 10.5, 15 mM hydrogen peroxide on the residual activity rate of rice peroxidase and Colprinus cinereus peroxidase Orange II (CI Acid Orange 7: Kanto) The effect of inactivation treatment on the residual activity rate was examined by a bleaching reaction using the following as a substrate.

2.5 mL of 0.1 M Tris-HCl buffer solution (pH 10.5) containing 0.06 mM Orange II was added into the cell of Hitachi UV-Vis spectrophotometer U-2010 (optical path 1 cm) and preincubated for 1 minute. . A 0.1 M Tris-HCl buffer solution (pH 10.5) containing 340 nM peroxidase and 15 mM hydrogen peroxide was co-mixed and stored for 20 minutes, and the reaction was started. The decrease value of the value of 485 nm, which is the maximum absorption wavelength of II, was determined. The decrease value one minute after the start of the reaction was divided by the decrease value when the reaction was similarly performed at a storage time of 0 minutes, and a value obtained by multiplying this by 100 was obtained as the residual activity rate (%). The results are shown in the following (Table 1). As rice-derived peroxidase, rice bran-derived peroxidase (product number K0310100: purity RZ> 1.2) sold by Funakoshi Co., Ltd. was used. The molar extinction coefficient of both enzymes was 1.02 × 10 ⁵ mol ⁻¹ dm ³ cm ⁻¹ (403 nm), similar to horseradish. For Orange II, a reagent manufactured by Wako Pure Chemical Industries, Ltd. was purified by a recrystallization method, and its purity was confirmed by using a paper distribution chromatogram and a UV spectrum. The results are shown in the following (Table 1).

  That is, it was found that rice-derived peroxidase exhibits a residual activity rate of 55% even after 20 minutes of inactivation treatment at 50 ° C., pH 10.5, and 15 mM hydrogen peroxide.

[Example 2] Effect of inactivation treatment at 50 ° C, pH 10.5, 15 mM hydrogen peroxide for 20 minutes on rate constants of rice-derived peroxidase and colprinus cinereus-derived peroxidase of Hitachi UV-Vis spectrophotometer U-2010 In the cell (optical path 1 cm), 2.5 mL of 0.1 M Tris-HCl buffer solution (pH 10.5) containing 0.06 mM Orange II was added and preincubated for 1 minute. The reaction was started by injecting 0.25 mL of 340 nM peroxidase and 0.25 mL of 15 mM hydrogen peroxide into 0.1 M Tris-HCl buffer solution (pH 10.5), and the maximum absorption wavelength of Orange II was 485 nm at 20 ° C. The change in absorbance over time was measured. Based on the following formula, the reaction rate constant before the inactivation treatment was determined. Similarly, 2.5 mL of 0.1 M Tris-HCl buffer solution (pH 10.5) containing 0.06 mM Orange II is added to the cell (optical path 1 cm) of Hitachi UV-Vis spectrophotometer U-2010 for 1 minute. Pre-incubated. The reaction was started by injecting 0.1M Tris-HCl buffer solution (pH 10.5) into which 340 nM peroxidase and 15 mM hydrogen peroxide were co-mixed and stored at 50 ° C. for 20 minutes. The change in absorbance over time at 20 ° C. at an absorption wavelength of 485 nm was measured. The reaction rate constant after inactivation treatment was determined based on the following formula.
As a result of analyzing the reaction rate equation from the fading curve of Orange II, it was found that the fading reaction proceeds in a pseudo first order reaction in any case. Therefore, the following equation (1) (Equation 1)

Here, k _t (min ⁻¹ ) is a pseudo first-order rate constant, C ₀ is the initial concentration of Orange II, and C _t is the concentration of Orange II at the reaction time t. From pseudo-first order kinetics shown) to determine the relationship between the rate constant k _t using the value of 30 seconds after the start of the reaction with hydrogen peroxide concentration in bleaching reactions Orange II. The results are shown in the following (Table 2).

[Example 3]
(Relationship between storage time and fading rate of rice-derived peroxidase and various peroxidases at 20 ° C., pH 10.5, 0.75 mM hydrogen peroxide)
2.5 mL of 0.1 M Tris-HCl buffer solution (pH 10.5) containing 0.06 mM Orange II was added into the cell of Hitachi UV-Vis spectrophotometer U-2010 (optical path 1 cm) and preincubated for 1 minute. . A reaction was started by injecting 5 mL of 680 nM peroxidase and 5 mL of 1.5 mM hydrogen peroxide solution which were mixed and stored for an arbitrary time. The value of 485 nm 30 minutes after the start of the reaction divided by the value of 485 nm when reacted with purified water instead of the hydrogen peroxide solution was multiplied by 100 times to obtain the color fading rate (%). The results are shown in the following (Table 3).

  That is, under the storage conditions of 20 ° C., pH 10.5, and 0.75 mM hydrogen peroxide, horseradish-derived peroxidase had a fading rate reduced to about 60% by storage processing for 150 minutes. The fading rate of was maintained around 85% and showed high stability. It was shown that rice-derived peroxidase is stable in the presence of 0.75 mM hydrogen peroxide, one of the detergent conditions, even after long-term treatment.

[Example 4]
(Relationship between storage time and residual activity of rice-derived peroxidase at 50 ° C., pH 10.5, 15 mM hydrogen peroxide)
An accelerated storage test when peroxidase was present as a solution composition coexisting with hydrogen peroxide and a stability evaluation under one of the washing conditions were performed.

  2.5 mL of 0.1 M Tris-HCl buffer solution (pH 10.5) containing 0.06 mM Orange II was added into the cell of Hitachi UV-Vis spectrophotometer U-2010 (optical path 1 cm) and preincubated for 1 minute. . Here, 5 mL of 680 nM peroxidase and 5 mL of 30 mM hydrogen peroxide were co-mixed at 50 ° C. and pH 10.5, and the mixture stored for an arbitrary period of time was injected to start the reaction at 50 ° C. The decrease in value was determined. Divide the decrease when reacting with a sample that has been co-mixed and stored for an arbitrary time by the decrease when reacting with a sample with a storage time of 0 minutes, and multiply this by 100 to obtain the residual activity rate (%) Asked. The results are shown in the following (Table 4).

  That is, when rice-derived peroxidase is stored at 50 ° C., pH 10.5, and 15 mM hydrogen peroxide, it has a high stability of 55% at a storage time of 20 minutes and a residual activity of 10% or more over a storage time of 20 minutes to 100 minutes. Indicated

[Example 5]
(Relationship between storage time and residual activity rate of rice-derived peroxidase at 50 ° C., pH 10.5, 375 mM hydrogen peroxide)
An accelerated storage test when peroxidase was present as a solution composition coexisting with hydrogen peroxide and a stability evaluation under one of the washing conditions were performed.

  2.5 mL of 0.1 M Tris-HCl buffer solution (pH 10.5) containing 0.06 mM Orange II was added into the cell of Hitachi UV-Vis spectrophotometer U-2010 (optical path 1 cm) and preincubated for 1 minute. . Here, 5 mL of 680 nM peroxidase and 5 mL of 750 mM hydrogen peroxide were co-mixed at 50 ° C. and pH 10.5, and the one stored for an arbitrary period of time was injected to start the reaction at 50 ° C. The decrease in value was determined. Divide the value when reacting with a sample that has been co-mixed and stored for an arbitrary time by the decrease value when reacting a sample with a storage time of 0 minutes, and multiply this by 100 to obtain the residual activity rate (%) It was. The results are shown in the following (Table 5).

  That is, even if rice-derived peroxidase is stored at 50 ° C., pH 10.5, 375 mM hydrogen peroxide, the storage time is 79% at 5 minutes, 51% at 20 minutes, 22% at 40 minutes, and 120 minutes. It was found to have a high stability of 21% residual activity.

[Example 6]
(Relationship between storage time, rate constant, and fading rate of rice-derived peroxidase at 50 ° C., pH 10.5, 375 mM hydrogen peroxide)
An accelerated storage test when peroxidase was present as a composition coexisting with hydrogen peroxide and a stability evaluation under one of the washing conditions were performed.

  In the same manner as in Example 5, 2.5 mL of 0.1 M Tris-HCl buffer solution (pH 10.5) containing 0.06 mM Orange II was placed in the cell of Hitachi UV-Visible spectrophotometer U-2010 (optical path 1 cm). In addition, preincubated for 1 minute. Here, 5 mL of 680 nM peroxidase and 5 mL of 750 mM hydrogen peroxide were mixed at 50 ° C. and pH 10.5, mixed and stored for an arbitrary period of time, and the reaction was started at 50 ° C. The reaction rate constant Kt was determined from the value. Further, the color fading rate (%) was determined from the value of 485 nm immediately after the reaction and 30 minutes after the start of the reaction. The results are shown in Table 6 below.

  That is, rice-derived peroxidase showed a reaction rate constant of 0.05 even after 0.25 and 120 minutes after 20 minutes of storage at 50 ° C. and pH 10.5 and 375 mM hydrogen peroxide. The fading rate was 56% at a storage time of 20 minutes and 38% at 120 minutes. It was found that even in the presence of a high concentration of hydrogen peroxide, it has high storage stability and reactivity.

[Example 7]
(Confirmation of storage stability of peroxidase and hydrogen peroxide precursor mixture)
A storage stability test was performed using sodium percarbonate as the hydrogen peroxide precursor mixture. Sodium percarbonate 5 g and peroxidase 0.1 g were stirred and mixed with 20 g of a commercially available synthetic detergent containing no bleach (containing no peroxidase), placed in a 100 mL polyethylene container, and stored at 37 ° C. in the dark. One month later, this was dissolved in 0.1 M Tris-HCl buffer solution (pH 9.0) to prepare a sample.

  2.5 mL of 0.1 M Tris-HCl buffer solution (pH 9.0) containing 0.06 mM Orange II was added into the cell of Hitachi UV-Vis spectrophotometer U-2010 (optical path 1 cm) and preincubated for 1 minute. . A reaction was started by injecting 0.25 mL of a sample and 0.25 mL of 9 mM hydrogen peroxide. The value of 485 nm 30 minutes after the start of the reaction divided by the value of 485 nm when the reaction was carried out using purified water instead of the hydrogen peroxide solution was multiplied by 100 times to obtain the color fading rate (%). The results are shown in the following (Table 7).

  That is, it showed a fading rate of 13% even after 1 month of storage, functioned stably as a detergent composition, and further storage stability could be expected by applying a known formulation of enzyme.

[Example 8] Examination of reaction rate constant of rice peroxidase under various hydrogen peroxide concentrations at 25 ° C. and pH 10.5 In the cell of Hitachi UV-Vis spectrophotometer U-2010 (optical path 1 cm), 0 2.5 mL of 0.1 M Tris-HCl buffer solution (pH 10.5) containing 0.06 mM Orange II was added and preincubated for 1 minute. The reaction was started by mixing 0.25 mL of 340 nM peroxidase and 0.25 mL of hydrogen peroxide of various concentrations into 0.1 M Tris-HCl buffer solution (pH 10.5), and the maximum absorption wavelength of Orange II was 485 nm. The reaction rate constant was determined from the change in absorbance after the start of the reaction.

  The results are shown in (Table 8). The hydrogen peroxide concentration shown here represents the hydrogen peroxide concentration at the start of the reaction. That is, it was shown that the hydrogen peroxide concentration showed a reaction rate constant of 1.2 or more from 0.75 mM to 75 mM, and even when it was 1 M or less, it had a reaction rate of 0.3 or more.

[Example 9] Effect of preventing dye transfer to test cloth The effect of preventing rice-derived peroxidase from transferring to a test cloth was examined in a system using Orange II as a model fiber dye.

  After removing water-soluble components in a 1: 1 volume ratio methanol-water mixed solution, the experimental nylon white fabric of the clothing life study group was air-dried, and then the oil-based components were removed with benzene and air-dried. This was used as a test cloth (white cloth, the same applies hereinafter).

  Put 0.2 g of test cloth into a vial, and add 9.5 mL of Carmody buffer solution (pH 10.5) containing rice peroxidase (360 nM) and hydrogen peroxide (15 mM) so that the final bath ratio is 1:50. In addition, stirring was started at 50 ° C. Next, 0.5 mL of 1 mM orange II aqueous solution was added to the vial and reacted at 50 ° C. for 30 minutes. After the reaction, the taken out test cloth was air-dried. Subsequently, the color difference (ΔE) was measured using a Minolta color difference meter CR-300 in order to express the degree of coloration of the orange II test cloth after the dye transfer prevention experiment.

  The color difference (ΔE) here is defined in order to quantitatively display the color sensation instead of the three attributes of hue, lightness, and saturation of the sensory colorimetry by the International Commission on Illumination (CIE). The numerical value being displayed. That is, L, a, and b which are parameters of the color difference (ΔE) are measured using a color difference meter, and ΔE can be calculated by Hunter's color difference formula (Equation 2) shown below.

  Further, as a comparative example, the same study was performed even in a system to which no rice-derived peroxidase was added. Furthermore, the case where the alkylbenzene sulfonate (LAS: anionic surfactant) was added to said system as a surfactant so that the final concentration might each be 7.35 mM was also examined. Table 9 below summarizes the results of each test section.

  As shown below, the addition of rice peroxidase prevented orange II from being transferred to the test cloth under the conditions of 50 ° C., pH 10.5, and hydrogen peroxide concentration of 15 mM. Further, the effect of preventing dye transfer was confirmed even in the presence of LAS.

  INDUSTRIAL APPLICABILITY The present invention is useful as a method for preventing dye migration in the washing or rinsing process, and performing wastewater treatment in the textile industry, etc. involving bleaching of fiber dye in a solution or dispersion, and a composition used in the method.

Claims (10)

  A method for preventing migration of fiber dye from one dyed cloth to another in the case of washing and / or rinsing together a plurality of cloths including cloth dyed in washing liquid, comprising: A method comprising causing a rice peroxidase having hydrogen peroxide resistance to act on a liquid in the presence of hydrogen peroxide.   A method for bleaching a fiber dye in a solution or dispersion, wherein the peroxidase derived from rice having hydrogen peroxide resistance is allowed to act on the solution or dispersion in the presence of hydrogen peroxide.   The method according to any one of claims 1 and 2, wherein the concentration of hydrogen peroxide is 0.001 mM or more and 1.0 M or less.   A method comprising oxidizing rice or a hydrogen donor by allowing rice peroxidase to act in a hydrogen peroxide concentration range of 0.001 mM to 1.0 M.   The method according to any one of claims 1, 2, 3, or 4, wherein the rice peroxidase is a rice bran peroxidase.   Hydrogen peroxide is a hydrogen peroxide precursor, a coexisting system of a bleach activator and a hydrogen peroxide precursor that can generate hydrogen peroxide by acting on the hydrogen peroxide precursor, and an enzyme that can generate hydrogen peroxide 6. A method according to any one of claims 1, 2, 3, 4, or 5, wherein the method is provided by means of at least one of the systems. .   A detergent composition or a bleaching composition containing a rice peroxidase having hydrogen peroxide resistance.   8. The composition according to claim 7, wherein the rice peroxidase is a rice bran-derived peroxidase.   The composition according to claim 7 or 8, wherein the concentration of hydrogen peroxide is 0.001 mM to 1 M when used as a detergent or a bleaching agent.   Hydrogen peroxide is a hydrogen peroxide precursor, a coexisting system of a bleach activator and a hydrogen peroxide precursor that can generate hydrogen peroxide by acting on the hydrogen peroxide precursor, and an enzyme that can generate hydrogen peroxide 10. A composition according to any one of claims 7, 8, or 9, characterized in that it is supplied by means of at least one of the systems.