JP2009124976A - Fluorescent dye-decomposing agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescent dye-decomposing agent high in safety and excellent in a fluorescent dye-decomposing ability, and to provide a method for decomposing a fluorescent dye. <P>SOLUTION: Provided is the fluorescent dye-decomposing agent for decomposing a fluorescent dye, containing manganese peroxidase as an active ingredient. Also, provided is the method for decomposing the fluorescent dye, comprising a process for reacting glucose oxidase with glucose in a solution containing the fluorescent dye to produce hydrogen peroxide, and a process for adding manganese peroxidase and divalent manganese ion to the solution to react and decompose the fluorescent dye in the solution. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a fluorescent dye decomposing agent. In particular, it relates to a fluorescent dye decomposing agent used in the environment and food environment fields.

  In recent years, problems with fluorescent dyes leaking from recycled paper are becoming apparent. Waste paper pulp (recycled pulp) uses waste paper such as so-called white stencil paper, high-quality paper, recycled paper, and newsprint paper, and most of the used paper pulp is reused as white stencil paper. For this reason, fluorescent dyes are used in the process for producing recycled pulp in order to make the recycled paper obtained from used paper pulp appear white. However, since the fluorescent dye has a chemical structure similar to that of harmful substances and is suspected to be carcinogenic, the safety of recycled paper containing the fluorescent dye is suspected. In particular, in the food field, it is forbidden to use recycled paper containing fluorescent dyes for applications such as food packaging paper. According to the Food Sanitation Law, fluorescent dyes should not be detected from paper that may come into contact with food.

  Therefore, virgin pulp is often used instead of recycled pulp for food packaging paper. However, due to cost problems, some waste paper pulp is actually used in confectionery boxes, pizza wrapping paper, and paper boxes for French fries used in fast food stores.

  For the above reasons, development of a method for removing fluorescent dyes in waste paper pulp is desired. Conventional methods for removing fluorescent dyes include reprocessing waste paper pulp directly with alkali or acid to decompose fluorescent dyes, applying chemicals to the surface of paper to prevent leakage of fluorescent dyes, stretching films, etc. The method (patent document 1) etc. are performed. In addition, chlorine-based fluorescent dye decomposing agents are also used (Patent Documents 2 to 5).

JP 2003-128033 A JP-A-62-97993 Japanese Patent No. 2116449 Japanese Patent No. 698365 Japanese Patent No. 3118087 JP 2002-27971 A Japanese National Patent Publication No. 11-508136 JP 2002-69881 A JP 2002-18480 A Modification of Pulp Fiber by Lignin-Degrading Enzyme System-Effect of MnP Treatment on DIP Pulp Fiber (Tokushu Paper Co., Ltd .; Tsuchikawa et al.)

  However, when the waste paper pulp is directly treated with alkali or acid to decompose the fluorescent dye, there is a problem that the waste paper pulp is deteriorated because the waste paper pulp is decomposed during the treatment. In addition, any of the methods such as coating the surface of the paper with a drug or stretching a film has a problem that the cost increases. In addition, although the chlorine-based fluorescent dye decomposing agent is excellent in the ability to decompose fluorescent dyes, it has a problem that it is very toxic. The handling of this chemical requires skill even for operators, and strict handling is required. Furthermore, chlorine-based fluorescent dye decomposing agents still have problems in wastewater treatment. That is, when wastewater in which unreacted chemicals remain is supplied to a wastewater treatment facility, microorganisms in the activated sludge treatment tank may be killed, and as a result, wastewater treatment cannot be performed.

  The present invention has been made in view of the above-described problems of the prior art, and an object of the present invention is to provide a fluorescent dye decomposing agent having high safety and excellent decomposing ability of fluorescent dyes.

  As a result of intensive studies, the present inventors have found that manganese peroxidase can decompose fluorescent dyes. Furthermore, the present inventor has found that manganese peroxidase has an excellent fluorescent dye decomposition effect and high safety. Based on this knowledge, the present inventor has made it possible to provide a fluorescent dye decomposing agent that is highly safe and excellent in decomposing ability of the fluorescent dye, and has completed the present invention.

  That is, the present invention provides a fluorescent dye decomposing agent that decomposes a fluorescent dye using manganese peroxidase as an active ingredient.

  Several functions have already been reported for the manganese peroxidase used in the present invention. That is, an action of decomposing a hardly decomposable halogenated carbohydrate compound such as trichlorethylene (Patent Document 6), an action of decomposing a dye by using it together with a mediator for enhancing the activity of the enzyme (Patent Document 7), pulp These include the action of decomposing lignin in the fiber (Patent Document 8), the decomposition of hardly decomposable substances such as dioxins in the waste water (Patent Document 9), and the like. In addition, at the lignin debate (1999), a method for treating waste paper pulp using manganese peroxidase was announced, and it was shown that bleaching based on delignification is possible, among which direct decomposition of fluorescent dyes Is not considered (Non-Patent Document 1). Thus, although several functions have been clarified, a direct fluorescent dye degrading action by manganese peroxidase has not been reported so far, and the fluorescent dye degrading action has been found for the first time this time.

  Manganese peroxidase used in the present invention is isolated from many microorganisms and produces trivalent manganese ions from divalent manganese ions under mildly acidic conditions. These trivalent manganese ions can decompose hardly decomposable phenolic compounds and pigments using their oxidizing power. Enzymes do not directly decompose phenolic compounds or pigments, but manganese ions with low molecular weight oxidize target compounds, so it is relatively difficult for large molecular weight enzymes to enter easily in fibers or many compounds that bind loosely. Trivalent manganese can also enter large particles. Therefore, the decomposition efficiency of the target compound is very high.

  Therefore, the fluorescent dye decomposing agent of the present invention can efficiently decompose the fluorescent dye in the reaction solution. Further, even when manganese peroxidase itself is in contact with the skin, the corrosiveness is very low, and it is only necessary to wash away with water. Therefore, the fluorescent dye decomposing agent of the present invention is extremely safer than the alkali, acid and chlorine based fluorescent decomposing agents conventionally used for decomposing fluorescent dyes.

The manganese peroxidase used in the present invention is preferably a manganese peroxidase derived from an edible basidiomycete.
By using manganese peroxidase derived from edible basidiomycetes as an active ingredient, safety to the human body and the like can be further enhanced.

The fluorescent dye in the present invention is preferably umbelliferone and / or a stilbene fluorescent brightener, and more preferably a stilbene fluorescent brightener.
The fluorescent dye decomposing agent of the present invention can efficiently decompose umbelliferone and / or a stilbene-based fluorescent brightening agent. Further, in the reaction for 30 minutes, umbelliferone decomposes 50% or more, and stilbene fluorescent whitening agent decomposes about 90%. Therefore, the fluorescent dye decomposing agent of the present invention is excellent in practicality.

  The present invention also includes a step of reacting glucose oxidase and glucose in a solution containing a fluorescent dye to produce hydrogen peroxide, adding manganese peroxidase and divalent manganese ions to the solution, and adding the fluorescent dye in the solution. And a method for decomposing the fluorescent dye, and a method for decomposing the fluorescent dye. The manganese peroxidase used in the present invention is preferably a manganese peroxidase derived from an edible basidiomycete. The fluorescent dye in the present invention is preferably umbelliferone and / or a stilbene fluorescent brightener, and more preferably a stilbene fluorescent brightener.

  According to the method for decomposing a fluorescent dye of the present invention, the fluorescent dye in a solution can be efficiently decomposed. In addition, the method for decomposing a fluorescent dye of the present invention is highly safe. Therefore, the method for decomposing a fluorescent dye of the present invention can be applied to pulp production, can be used for wastewater treatment of wastewater containing a fluorescent dye, and has an environmental purification action. Manganese peroxidase itself does not adversely affect microorganisms in the activated sludge, and there is no problem even if it is used in a normal drainage line. Therefore, the method for decomposing the fluorescent dye of the present invention is excellent in terms of cost and equipment.

  Furthermore, the present invention provides a method for producing regenerated pulp in which the method for decomposing a fluorescent dye of the present invention is performed in the method for producing regenerated pulp using waste paper containing a fluorescent dye as a raw material, and the fluorescent dye in the pulp is decomposed. To do. In addition, a regenerated pulp obtained by the above-described method for producing regenerated pulp is provided. The recycled pulp is preferably used as a food packaging paper.

  According to the method for producing regenerated pulp of the present invention, since the fluorescent dye contained in the waste paper that is the raw material of the regenerated pulp can be decomposed, the regenerated pulp in which the fluorescent dye is decomposed efficiently and safely is produced. Can do. Moreover, the regenerated pulp in which the fluorescent dye was efficiently reduced can be provided. Since the fluorescent dye is efficiently reduced, leakage of the fluorescent dye from the recycled paper can be reduced, and a recycled pulp that is highly safe and suitable for use as a food packaging paper can be provided. .

  INDUSTRIAL APPLICABILITY According to the present invention, a fluorescent dye decomposing agent and a fluorescent dye decomposing method having high safety and excellent effects are provided. Moreover, the regenerated pulp in which fluorescent dye was reduced and the manufacturing method thereof can be provided. The present invention is effective in environmental purification and wastewater treatment, and is used for decomposing fluorescent dyes in any wastewater as long as it is wastewater from detergent factories that use fluorescent whitening agents, or other wastewater containing fluorescent dyes. Can do. It is excellent in terms of cost and equipment.

  Hereinafter, although the structure and preferred embodiment of this invention are explained in full detail, this invention is not limited by this.

  First, the fluorescent dye decomposing agent in this embodiment will be described. The fluorescent dye decomposing agent in this embodiment contains manganese peroxidase as an active ingredient.

  Manganese peroxidase is an enzyme that oxidizes divalent manganese ions to trivalent manganese ions. The manganese peroxidase used in the present invention requires hydrogen peroxide for the expression of activity, and the enzyme exhibits maximum activity in a weakly acidic region. In order to further develop the activity, a surfactant such as fatty acid or Tween 80 may be included in the reaction system. Manganese peroxidase may be any one as long as it is obtained from microorganisms such as white rot fungi and brown rot fungi belonging to basidiomycetes. Of these, edible basidiomycetes are preferred for safety reasons. It is also possible to obtain a large amount of manganese peroxidase by introducing the manganese peroxidase gene present in the genome of white rot fungi or brown rot fungus into another host and expressing it high. Specifically, a manganese peroxidase mass expression construct is prepared by preparing cDNA from mRNA encoding manganese peroxidase and connecting the cDNA downstream of the mass expression promoter. There is a method for preparing a large amount of manganese peroxidase by introducing this construct into a yeast such as Pichia pastoris. If the above manganese peroxidase can be obtained, the manufacturing method will not be specifically limited. In addition to the manganese peroxidase, the fluorescent dye decomposing agent in this embodiment may be provided with a divalent manganese ion, a hydrogen peroxide or hydrogen peroxide source, a buffer solution, etc., and prepared immediately before the start of the reaction. As such, it may be composed of a plurality of reactants (preferably, a reaction solution).

  Next, a method for decomposing the fluorescent dye in this embodiment will be described. The method for decomposing a fluorescent dye in the present embodiment includes a step of reacting glucose oxidase and glucose in a solution containing the fluorescent dye to generate hydrogen peroxide (hydrogen peroxide generating step), manganese peroxidase and divalent in the solution. And a step of decomposing the fluorescent dye by reacting with the fluorescent dye in the solution (fluorescent dye decomposition step). Manganese peroxidase used in the present embodiment may be the fluorescent dye decomposing agent described above.

  Since hydrogen peroxide is required when using manganese peroxidase, it is necessary to consider the hydrogen peroxide addition system. (1) When hydrogen peroxide is added directly to the reaction system, (2) When using glucose oxidase and generating hydrogen peroxide using glucose as a substrate, there are two methods. In particular, in the hydrogen peroxide generation step of the present embodiment, the method (2) is preferable in that hydrogen peroxide can be continuously supplied to the reaction system and the activity of manganese peroxidase is maintained. The concentration of hydrogen peroxide in the reaction system is preferably 1 mM or less, more preferably 0.05 mM or more and 0.5 mM or less. When the concentration of hydrogen peroxide in the reaction system is higher than 1 mM, the activity of manganese peroxidase may be reduced or the activity itself may be lost. Further, when the concentration of hydrogen peroxide in the reaction system is less than 0.05 mM, the amount of hydrogen peroxide involved in the reaction becomes insufficient, and the reaction may not proceed efficiently. In the addition method (1), it may be difficult to make the concentration of hydrogen peroxide uniform or to keep it within a certain amount or within a certain range. In this case, the activity of manganese peroxidase decreases or the activity itself is lost. Sometimes it breaks.

  In the next fluorescent dye decomposition step, the conditions for using manganese peroxidase are 30 ° C. to 70 ° C., preferably 37 ° C. to 60 ° C., more preferably 45 ° C. to 55 ° C. The reaction pH is pH 3.0 to 6.9, preferably pH 4.0 to 6.0, and more preferably pH 4.5 to 5.5. In the decomposition of the fluorescent dye, there is no problem if the manganese peroxidase to be used is added in an appropriate amount. However, in consideration of economy, the enzyme titer in the reaction system is 0.01 U / ml to 100 U / ml, preferably The range is 0.05 U / ml to 5 U / ml, more preferably 0.05 U / ml to 2 U / ml.

  When the fluorescent dye is decomposed, in order to increase the decomposition efficiency of the fluorescent dye in the pulp fiber, it can be used in combination with known lignin-degrading enzymes such as lignin peroxidase and laccase. Further, it can be used in combination with enzymes such as xylanase and amylase which are polysaccharide degrading enzymes.

  A fluorescent dye refers to a dye whose pigment is a fluorescent substance and is also referred to as a fluorescent agent. In particular, a fluorescent dye added to waste paper pulp or laundry detergent for the purpose of whitening effect by fluorescence is called fluorescent whitening agent. Some fluorescent dyes have similar chemical structures to harmful substances and are suspected to be carcinogenic, so the safety of fluorescent dyes is suspected. In addition, fluorescent dyes are generally very poorly degradable, and it is said that it takes more than two months to be completely decomposed in the environment. In other words, it is mixed with wastewater from the production of recycled pulp and discharged into the river and remains, causing environmental pollution.

  The present inventors have found that manganese peroxidase is excellent in two decomposition properties among fluorescent dyes: (1) a coumarin fluorescent dye such as umbelliferone and (2) a stilbene fluorescent brightener. It was. At present, there are many stilbene compounds as fluorescent brighteners used in waste paper and detergents, and manganese peroxidase can efficiently decompose the fluorescent brightener.

  A fluorescent dye decomposing agent containing manganese peroxidase as an active ingredient is very safe compared to a chlorine-based fluorescent dye decomposing agent. Manganese peroxidase itself is very corrosive even when it comes into contact with the skin, and it can be washed away with water. On the other hand, when a chlorine-based fluorescent dye decomposing agent comes into contact with the skin, it causes symptoms such as inflammation and wrinkles, and may require treatment by a doctor.

  When using a fluorescent dye decomposing agent comprising manganese peroxidase of the present invention as an active ingredient, an enzyme solution containing manganese peroxidase may be added directly to the reaction system, but manganese peroxidase is immobilized on a carrier and reacted with hydrogen peroxide. Only trivalent manganese ions produced in this manner may be used as a decomposing agent.

  The fluorescent dye degrading agent containing manganese peroxidase as an active ingredient of the present application may be waste paper pulp containing fluorescent dye, waste paper pulp waste water, waste water from detergent factories using fluorescent whitening agents, or waste water containing fluorescent dyes. For example, it can be used for decomposition of any wastewater fluorescent dye. Manganese peroxidase itself does not adversely affect microorganisms in the activated sludge, and there is no problem even if it is used in a normal drainage line.

  Finally, the manufacturing method of the regenerated pulp in this embodiment is demonstrated. The method for producing regenerated pulp in the present embodiment is a method for producing regenerated pulp that decomposes the fluorescent dye in the pulp by performing the method for decomposing the fluorescent dye in the method for producing regenerated pulp using the waste paper containing the fluorescent dye as a raw material. Is the method.

  Conventional methods for producing recycled pulp generally include a disaggregation process in which waste paper is disaggregated to obtain a pulp suspension, a coarse selection / selection process in which foreign matter in the pulp suspension is separated, and a printing ink is separated. Regenerated pulping is carried out through a deinking process that performs and a bleaching process that whitens the color of the regenerated pulp.

  In the method for producing regenerated pulp in the present embodiment, the above-described method for decomposing a fluorescent dye is performed instead of the conventional bleaching step. Thereby, the fluorescent dye contained in the waste paper which is the raw material of the recycled pulp can be decomposed efficiently and safely. Therefore, a regenerated pulp in which the contained fluorescent dye is reduced can be obtained. Since the fluorescent dye is efficiently reduced, leakage of the fluorescent dye from the recycled paper can be reduced, and a recycled pulp that is highly safe and suitable for use as a food packaging paper can be provided. .

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by this.

  First, preparation examples of manganese peroxidase of this example and measurement examples of enzyme activity and fluorescent dye decomposing activity are shown. Next, an example in which the resolution of the fluorescent dye using the fluorescent dye decomposing agent of this example was inspected is shown, and finally, an example in which the safety of the fluorescent dye decomposing agent of this example was inspected is shown.

<Preparation Example 1: Culture of white rot fungi>
The white rot fungus Phanerochaete sordida (ATCC 90872) was used as the strain used. An enzyme solution containing manganese peroxidase derived from white rot fungi was prepared from a culture solution of white rot fungi.

  The method for cultivating white rot fungi is as follows. First, a white rot fungus was pre-cultured on a potato dextrose agar medium at 30 ° C. for 3 days. Next, the cells were collected after pre-culture and homogenized with a mixer to inoculate the liquid culture medium for main culture. The liquid medium of the main culture is a liquid containing 10 g / l glucose, 0.22 g / l (1.2 mM) ammonium tartrate, 1.64 g / l (20 mM) sodium acetate, 1.0 g / l Tween 80, and a trace metal salt. It was a medium (pH 4.5), and 200 ml of liquid medium was used for a 500 ml Erlenmeyer flask. The main culture was performed under the following conditions. After inoculation of the cells, the cells were cultured with shaking at 30 ° C. and 150 rpm. On the third day of culture, veratryl alcohol was added to a final concentration of 1.25 g / l. Thereafter, while the inside of the flask head space was replaced with oxygen gas for about 1 hour every day, the culture was continued with shaking at 30 ° C. and 150 rpm. Finally, it was cultured for 7 days.

Then, an enzyme solution containing manganese peroxidase derived from white rot fungi was prepared from the obtained white rot fungus culture solution by the following method. After removing the cells with filter paper, the filtrate filtered through a membrane filter having a pore size of 0.2 μm was collected, and this was designated as enzyme solution 1.
Manganese peroxidase enzyme activity of enzyme solution 1 obtained from the culture solution on the last day of culture was 2.5 U / ml. The following method was used for measuring the enzyme activity.

<Method for Measuring Manganese Peroxidase Enzyme Activity>
The reaction solution used for measuring enzyme activity had the following composition. 1.0 mM 2,6-dimethoxyphenol, 1.0 mM manganese sulfate (II), 50 mM sodium malonate buffer (pH 4.5), 0.2 mM hydrogen peroxide solution. Prior to the start of the reaction, a reaction solution other than oxygen peroxide and 100 μl of enzyme solution were mixed in the cell and kept at 37 ° C. for 5 minutes. And hydrogen peroxide water was added so that it might become final concentration of 0.2 mM, the final amount of the liquid mixture was made into 1 ml, and reaction was started at 37 degreeC. The absorbance change at 469 nm based on the oxidation of 2,6-dimethoxyphenol by the produced Mn (III) -malonic acid complex was measured. The enzyme activity was calculated with a molar extinction coefficient of 49.6 mM ⁻¹ cm ⁻¹ . One unit [U] of the enzyme activity of manganese peroxidase was defined as the value at which 1 μmol of 2,6-dimethoxyphenol oxide was produced per minute.

<Preparation Example 2: Large-scale preparation of manganese peroxidase by genetic recombination>
(1) Cloning of Manganese Peroxidase Gene Manganese peroxidase cDNA of white-rot basidiomycete Phanerochaete chrysosporum (ATCC 24725) was cloned by the following method. For culturing cells, CF11 (cotton-derived cellulose fiber) was used as a substrate, the substrate concentration was 2%, urea was added as a nitrogen source, 0.06%, and ammonium hydrogenphosphate was added as 0.026%. After inoculating the above-mentioned liquid culture of the main culture, the cells were cultured with shaking at 37 ° C. and 150 rpm for 4 days. After the culture, the cells were collected by filtration with a glass filter. The obtained microbial cells were freeze-ground with liquid nitrogen and used as a material for mRNA extraction. First, E.I. Z. N. A. Total RNA was extracted using a (trademark) Fungal RNA extraction kit (Omega Bio-Tek). Next, mRNA was purified using Oligotex (trademark) -dt30 <Super> mRNA Purification kit (manufactured by Takara Bio Inc.). The reverse transcription reaction from the obtained mRNA to cDNA was performed using RiverTra Ace (Toyobo) and oligo (dT) 30 primer. Using the obtained cDNA as a template, PCR was performed using specific primers of the manganese peroxidase 5 gene, and the full-length manganese peroxidase cDNA was cloned. The gene-specific primer for manganese peroxidase 5 was designed based on the genome information of Phanerochaete chrysosporium. Specific primer sequences are as follows. Foward: 5′-CGACAGACAG GGCTCGCCGTG TGCG-3 ′ (SEQ ID NO: 1) and Reverse: 5′-TCAAGCCGGGG CCGTCGAA-3 ′ (SEQ ID NO: 2).

(2) Construction of Manganese Peroxidase Expression Construct The obtained cDNA was subjected to restriction enzyme treatment using EcoRI and NotI (manufactured by Takara Bio), and DNA ligation Kit (manufactured by Takara Bio) as a ligation kit, and Pichia pastoris (Pichia pastoris) Ligation was performed with pPICZαA (manufactured by Invitrogen), which is an expression vector. Then, using this reaction solution, it was transformed into competent cell JMI09 (manufactured by Takara Bio Inc.) and cloned to obtain a manganese peroxidase expression construct (MnP5 / pPICZαA).

(3) Manganese peroxidase protein expression Manganese peroxidase expression construct was subjected to restriction enzyme treatment with Bpu1102I (manufactured by Takara Bio Inc.), and the resulting double-stranded linear DNA was transformed into Pichia pastoris KM-71 strain by electroporation. Introduced. Selection of transformants was performed using antibiotic resistance (Zeocin resistance) as an index.

  The above transformant was cultured in a YPG medium (containing 25 ug / ml Zeocin), and the cells were collected. Next, after shaking culture at 27 ° C. and 150 rpm for 3 days using YP medium containing Hemin (1000 μmol) and methanol (1%) as a carbon source, the extracellular solution was centrifuged at 5000 rpm for 20 minutes. After separation, the supernatant was recovered and used as enzyme solution 2. The manganese peroxidase activity of this enzyme solution was 1 U / ml.

<Example 1: Decomposition of umbelliferone>
Using the enzyme solution 1 and the enzyme solution 2 obtained in Preparation Example 1 and Preparation Example 2, an experiment for examining the resolution of the fluorescent dye was performed. The substrate used was the fluorescent dye umbelliferone.
4-0-methylumbelliferone (manufactured by Sigma) was dissolved in 50 mM malonate buffer (pH 4.5) so as to be 100 μmol / l, and this was used as a substrate solution. As the enzyme stock solution, the enzyme solutions 1 and 2 prepared to have an enzyme activity of 1 U / ml were used. A 50 mM malonate buffer solution (pH 4.5) was used for dilution preparation of the enzyme titer of the enzyme solution.

  The reaction system of Example 1 was prepared using 50 mM malonate buffer (pH 4.5) so that the final volume was 200 μl. In the reaction system, manganese sulfate was added to a final concentration of 0.5 mM, and hydrogen peroxide was added to a final concentration of 0.5 mM in addition to the substrate solution (final concentration of 0.1 mM). Moreover, the enzyme solution was added to the reaction system so that it might become 0.05 U / ml. For the control experiment, the heat-inactivated enzyme solution 1 was used. The reaction was carried out at 37 ° C.

  The fluorescence intensity was measured using Fluoroscan Ascent manufactured by Dainippon Sumitomo Pharma Co., Ltd. at an excitation wavelength of 355 nm and a measurement wavelength of 450 nm. After enzyme addition (after the start of the reaction), the fluorescence intensity at 0 min, 15 min and 30 min was measured.

<Result>
The results of Example 1 are shown in Table 1. Table 1 shows the change in fluorescence intensity associated with the decomposition of the fluorescent dye by manganese peroxidase. From the results of Example 1, it was revealed that enzyme solutions 1 and 2 having manganese peroxidase enzyme activity have 4-0-methylumbelliferone resolution. It was also found that 47% of 4-0-methylumbelliferone was decomposed 30 minutes after the start of the reaction.

<Example 2: Decomposition of stilbene brightener>
Example 2 was carried out in the same manner as Example 1 except for the fluorescent material used for the substrate. As the substrate, Kyaphor NW (manufactured by Nippon Kayaku), which is a stilbene brightener, was used, and the substrate concentration in the reaction system was 0.001%.

<Result>
The results of Example 2 are shown in Table 2. Table 2 shows changes in fluorescence intensity accompanying the decomposition of the fluorescent dye by manganese peroxidase. From the results of Example 2, it was revealed that enzyme solutions 1 and 2 having manganese peroxidase enzyme activity have Kayphor NW resolution. It was also found that 90% Kyaphor NW was decomposed 30 minutes after the start of the reaction.

  From the results of Examples 1 and 2, it was found that manganese peroxidase can decompose fluorescent dyes and fluorescent brighteners. Further, under the conditions of this example (reaction time 30 min), manganese peroxidase decomposes umbelliferone and stilbene-based brighteners by less than half and 90%, respectively, so that enzyme solution 1 and enzyme solution 2 in this example The reaction system was found to be practical and efficient. Patent Document 9 discloses the decomposition of dioxins by microorganisms. In this document, it takes one month to decompose 90% of dioxins. From this, it can be seen that manganese peroxidase is remarkably excellent in the decomposition characteristics of coumarin fluorescent dyes and stilbene brighteners. Therefore, by treating with manganese peroxidase, the fluorescent material can be efficiently and practically decomposed in waste paper pulp containing fluorescent dyes and fluorescent whitening agents and wastewater during pulp production.

<Safety test>
In order to confirm the safety of manganese peroxidase, a “skin irritation test” and an “acute oral toxicity test” were performed by the following methods.

<Skin irritation test>
Enzyme solution 1 and enzyme solution 2 obtained by Preparation Example 1 and Preparation Example 2 (1.0 U / ml, 100 ul, respectively) were respectively removed from C3H mice (male, 6 weeks of age, 10 mice in each group) It was continuously applied to the back of Nippon Charles River Co., Ltd. (once / day for about 1 month). Abnormalities such as erythema, edema and inflammation were not particularly observed on the back of the mice during the application period and 2 weeks after the application was completed. In addition, no significant difference (P <0.05) was observed in the body weight transition as compared with the control group (water application group).

  Enzyme Solution 1 and Enzyme Solution 2 (each 1.0 U / ml) were each treated with a gastric sonde on ICR mice (male, 6 weeks old, 10 per group, manufactured by Charles River Japan) after hair removal. And orally administered (dosage 5 g / mouse body weight 1 kg). There were no deaths until 2 weeks after administration. In addition, there was no significant difference (P <0.05) in the body weight transition compared to the control group (water administration group).

  The fluorescent dye decomposing agent containing manganese peroxidase obtained as an active ingredient in the present invention is highly safe and can be used in the fields of environmental purification, wastewater treatment, and pulp production.

Claims (9)

  A fluorescent dye decomposing agent that decomposes fluorescent dyes with manganese peroxidase as an active ingredient.   The fluorescent dye decomposing agent according to claim 1, wherein the manganese peroxidase is a manganese peroxidase derived from an edible basidiomycete.   The fluorescent dye decomposing agent according to claim 1 or 2, wherein the fluorescent dye is a stilbene fluorescent whitening agent. Reacting glucose oxidase and glucose in a solution containing a fluorescent dye to produce hydrogen peroxide;
Adding manganese peroxidase and divalent manganese ions to the solution and reacting with the fluorescent dye in the solution to decompose the fluorescent dye;
A method for decomposing a fluorescent dye comprising:   The method for decomposing a fluorescent dye according to claim 4, wherein the manganese peroxidase is a manganese peroxidase derived from an edible basidiomycete.   The method for decomposing a fluorescent dye according to claim 4 or 5, wherein the fluorescent dye is a stilbene fluorescent whitening agent. In a method for producing recycled pulp using waste paper containing a fluorescent dye as a raw material,
The manufacturing method of the regenerated pulp which performs the decomposition | disassembly method of the fluorescent dye as described in any one of Claims 4-6, and decomposes | disassembles the fluorescent dye in a pulp.   Regenerated pulp obtained by the production method according to claim 7.   The regenerated pulp according to claim 8, wherein the regenerated pulp is used as a food packaging paper.