WO1998032871A1

WO1998032871A1 - Method for polymerizing phenolic compounds or the like and use thereof

Info

Publication number: WO1998032871A1
Application number: PCT/JP1998/000172
Authority: WO
Inventors: Takashi Echigo; Tadashi Yoneda; Hirobumi Aoki; Ritsuko Ohno
Original assignee: Showa Denko K.K.
Priority date: 1997-01-22
Filing date: 1998-01-19
Publication date: 1998-07-30
Also published as: AU5497498A

Abstract

A method for polymerizing phenolic compounds or the like with a polyphenol oxidase having a optimal reaction pH on the alkaline side; compositions prepared by using the above method, such as a thickener, a stabilizer, a coagulant, an emulsifier, a dispersant, a water retaining agent, a humidity conditioning agent, an antioxidant, an adhesive, a dye, a coating agent, a rust preventive, a flame retardant, a petroleum recovering agent, a soil conditioner, a stabilizer for a surface soil sprayed with a seed, a deodorizer, a deodorant, an agricultural chemical spreading agent, a deoxydizer, a preservative, a disinfectant, an antimicrobial agent, a viral infection inhibitor, an organism deposition inhibitor, an organism repellant, an insecticide, an insect repellant, a cataplasm, an ink base, a concrete admixture, and a wood treatment; processes for preparing the above compositions; a method for treating soil; a method for treating a porous article; and a treated porous article and a process for preparing the same.

Description

Description Method for polymerizing phenolic compounds and uses thereof

The present invention relates to a method for utilizing a novel polyphenol oxidase having an optimum reaction pH on the side of the reaction force.

More specifically, the present invention relates to a novel polyphenol oxidase having an optimum reaction pH on the side of pH 8 near phenol, a phenol compound, an alkoxyl group-containing aromatic compound, a halogenated phenol compound, and a quinone compound. Or a method of polymerizing these compounds by acting on an aromatic amine compound, and rapidly converting a phenolic compound, an alkoxyl group-containing aromatic compound, a halogenated phenolic compound, a quinone compound or an aromatic amine compound into a polymer. Thickeners, stabilizers, flocculants, emulsifiers, dispersants, water retention agents, humidifiers, antioxidants, adhesives, dyes, paints, paints, etc. Agents, flame retardants, oil recovery agents, soil modifiers, seed spray topsoil stabilizers, deodorants, deodorants, pesticide spreading agents, oxygen absorbers, preservatives, disinfectants Manufacture of compositions such as antibacterial agents, virus infection inhibitors, biofouling inhibitors, biological repellents, insecticides, insect repellents, cataplasms, ink bases, concrete admixtures, wood treatment agents, etc., and these various compositions Methods, soil treatment methods, and even metal sintered compacts, metal products, alloys, die-cast products, ceramics, bricks, concrete, wood, wood-based processed materials, fir, rush, straw, bamboo, or synthetic resin The present invention relates to a method for treating a porous article such as a foam. Background art

Heretofore, the polyphenol oxidizing effect of polyphenol oxidase perlaccase produced by filamentous fungi such as basidiomycetes and incomplete fungi has been known. However, conventional polyphenol oxidases have significantly lower activity on the alkaline side. Therefore, the actual use was limited.

As natural products having polyphenol in the structural part, plant pigments and lignins such as flavonoids, xanthones, and melanin are known, and polyphenol oxidase has an oxidizing effect on these natural products. In addition, polyphenol oxidase can be used as a reaction substrate even for dichlorophenol and trichloromouth phenol, for which toxicity is a problem. Therefore, polyphenol oxidase is also useful in treating wastewater containing these natural and non-natural products. However, the conventional enzyme has an optimal reaction pH from acidic pH to neutral pH, and it is practically difficult to use it in an alkaline pH range, which is a factor in the industrial use of polyphenol oxidase. This was a factor that narrowed the range of use.

It is also known that phenolic compounds and the like can be polymerized using enzymes such as laccase-polyphenoloxidase produced by basidiomycetes and incomplete fungi (Journal of Biotechnology, 13, 229). -241, 1990, etc.). However, since laccase-polyphenoloxidase produced by fungi has an optimal reaction pH in the acidic region, in order to catalyze the polymerization reaction using these enzymes, it is necessary to convert the reaction from acidic to acidic. The reaction had to be carried out in the neutral pH range, and the rate of the polymerization reaction was not high enough. In addition, many of the natural organic compounds to which these enzymes can react are polyphenol compounds, and although the solubility of these polyphenol compounds decreases in the acidic to neutral pH range, the optimal reaction of the enzyme p Since H is in the acidic region, the reaction must be carried out in the acidic to neutral pH range, and there has been a drawback that high-concentration polyolefin compounds cannot be efficiently polymerized. In addition, despite the fact that many polyphenol compounds accelerate their autoxidation in the alkaline pH range, they have conventionally performed enzymatic oxidative polymerization in the acidic to neutral pH range. There was a disadvantage that it could not be used effectively in a polymerization reaction.

Conventionally, various enzymatic polymerization methods using borihinol oxidase as a polymerization catalyst , W087-2939, JP-A-5-117591, JP-A-6-287516, JP-A-7-126354, JP-A-7-126377 and the like. However, these enzymatic polymerization methods involve polymerization reactions in solution or on solid surfaces and their use. In addition, a method of using a reaction of polymerizing a polyphenol substance such as urushiol with laccase as a polymerization catalyst for a coating agent or an adhesive has been used as lacquer since ancient times. Attempts have also been made to improve the water resistance of cardboard by applying an enzyme having a polyphenol oxidizing action such as laccase together with its substrate to the cardboard. However, these lacquer utilization methods and similar technologies are essentially polymerization reactions on the solid surface or solid bonding surface, and the additives to the reaction solution for the polymerization reaction are also colored. It was limited to paints and water-soluble polysaccharides that imitated natural lacquer, and the industrial use and application of lacquer-like reactions were limited.

Wood also consists of a first stage in which derivatized lignin is added to wood to prevent microbial corrosion, and a second stage in which a weakly acidic aqueous solution containing metal ions is impregnated in order to make the lignin water-insoluble. An impregnation method has been tried in JP-A-61-268729. However, in this method, the solubility of the complex of the lignin derivative and the metal ion in water is reduced, but the lignin derivative itself is not fixed as a water-insoluble substance. In addition, two types of processing agents must be switched and used. Injecting a CCA-based aqueous solution (chromium-copper-arsenic compound) that can be processed with one agent, which is currently mainly used as a wood preservative It is difficult to introduce it to other facilities. Therefore, there has been a demand for the development of a method of using a lignin derivative having a strong adhesion to wood and a method capable of treating with one agent.

Conventionally, various chemicals have been impregnated into porous articles to provide strength, abrasion resistance, weather resistance, fire resistance, flame retardancy, antibacterial properties, antiseptic properties, bactericidal properties, insect repellency, insecticidal properties, Virus, biorepellent, adhesive, sustained release of drug, coloring, dimensional stability, anti-cracking, humidity control, water absorption, water repellency, surface smoothness, biocompatibility, ion exchange, formaldehyde It is intended to impart or improve absorption, drug leaching prevention, or migration prevention of inorganic compounds to the surface of porous articles, but the injected drug leaches out For this reason, it is difficult to impart the above properties that are effective for a long period of time to porous articles, and there is a concern that the leached drug may affect the environment and the human body. There has been a need for a method of treating a porous article that has a long-lasting effect and is easy to treat.

Conventionally, for the purpose of modifying physical properties, imparting strength, manufacturing aggregates, imparting antibacterial properties and insect repellency, etc., sintered bodies such as bricks, wood, wood chips, wood flour, fiber, paper, or A method of impregnating or applying a thermosetting resin or the like to a porous article such as pulp has been implemented. However, since unreacted formaldehyde is contained in the thermosetting resin, there is concern about the effect on the human body, and the use of a compound that does not contain formaldehyde has been required. In addition, since heating at 80 to 200 ° C. is required for curing the thermosetting resin, there is a disadvantage that a special heating facility and energy for heating are required.

On the other hand, among animal and plant-derived polyphenol oxidases, those having an optimum pH in a high pH region of pH 8 or higher are known. Comp. Biochem. Physiol., 1992, 102B (4) 891 -896: Zhongguo Nongye Huaxue Huizhi, 1991, 29 (2), 177-185: Agric. Biol. Chem., 1991, 55 (1), 13-17). However, it is difficult to produce such polyphenoloxidase stably and inexpensively from animal and plant tissues, and microbial polyphenoloxidase has been desired for industrial use.

.

Purpose of the invention

An object of the present invention is to provide a novel enzyme-catalyzed method for polymerizing a phenol compound, an alkoxyl group-containing aromatic compound, a halogenated phenol compound, a quinone compound, or an aromatic amine compound. is there.

Another problem to be solved by the present invention is to reduce the practical enzymatic oxidation in the pH range by using polyphenol oxidase having an optimal reaction pH on the side of the enzyme. Achieved and expanded applications of polyphenoloxidase It is to contribute greatly. Disclosure of the invention

The present inventors have diligently searched for a wide range of microorganisms for extracellular products that catalyze the oxidation of polyphenolic substances at alkaline pH, and have found that the imperfect fungi spp.Stilbella and Sagenomella spp. Alternatively, the present inventors have found that a strain belonging to the genus Stachylidium produces an objective enzyme having an optimum reaction pH on the alkaline side near pH 8 outside the cells, thereby completing the present invention. .

In addition, the present inventors use these enzymes in an alkaline region, particularly in an alkaline region having a pH of 8 or more, to obtain a phenol compound, an alkoxyl group-containing aromatic compound, a halogenated phenol compound, a quinone compound, The present inventors have found that it is possible to efficiently achieve the polymerization of an aromatic amine compound, and have completed the present invention.

Further, the present inventors have developed a long-term effective strength, abrasion resistance, weather resistance, weather resistance, flame retardancy, antibacterial, antiseptic, bactericidal, insect repellent, insecticidal, antiviral, Bio-repellent, adhesive, sustained-release of drug, coloring, dimensional stability, anti-cracking, deodorant, deoxidizing, humidity-controlling, water-absorbing, water-repellent, surface smoothness, bio-affinity, ion exchange To develop a method for imparting or improving the properties, formaldehyde absorption, drug leaching prevention, or migration of inorganic compounds to the surface of porous materials in porous articles such as wood, intensive research has been conducted. went. Then, the polyphenol oxidase and the phenol compound, the alkoxyl group-containing aromatic compound, the halogenated phenol compound, the quinone compound, or the aromatic amine compound of the present invention, and the unsaturated compound and / or the drug are porous. It has been found that the object of the present invention can be achieved by impregnating or impregnating an article by applying pressure and / or depressurization, and performing a polymerization reaction in a porous article, thereby completing the present invention. .

That is, the present invention provides a polyphenol utilizing oxygen (air) as an oxidizing agent. Although oxidase is used, it is surprising that such an enzyme or enzyme system catalyzes an oxidation reaction or a polymerization reaction in an environment with a low oxygen supply rate inside a porous article. In particular, impregnation, which includes a decompression operation as part of the treatment process, is extremely effective as an operation for increasing the injection amount of the treatment liquid into porous articles that are difficult to impregnate. However, the concentration of dissolved oxygen in the processing solution and the processed product that has undergone the depressurization operation is low, which is disadvantageous for a catalytic reaction using oxygen as an oxidizing agent. However, surprisingly, it has been found that even in a processed product of a porous article that has been subjected to a decompression operation, a catalytic oxidation reaction and a polymerization reaction in the inside proceed.

The pressure injection method is also very effective as an operation to increase the injection amount of the processing solution into porous articles that are difficult to impregnate, but many enzymes are unstable to the pressure treatment. Are known. However, surprisingly, the polyphenol oxidase of the present invention maintains its catalytic activity even after the pressure injection treatment, and furthermore, in a low oxygen supply rate environment inside the porous article, the oxidation reaction, It has been found that it catalyzes the molecular reaction.

In addition, the purpose is to increase the treatment effect of the porous article, and to fix the drug with a phenolic compound, an alkoxyl group-containing aromatic compound, a halogenated phenol compound, a quinone compound, or an aromatic amine compound polymerized inside the porous article. Various fragrances, deodorants, antioxidants, flame retardants, antibacterials, preservatives, bactericides, insect repellents, antivirals, We conducted intensive research on a treatment method using a biorepellent. In addition, the polymerization reaction using the polyphenol oxidase of the present invention generally involves the immobilization or slowing down of many drugs including drugs that are thought to cause enzyme reaction inhibition or enzyme inactivation. They found that they could be released and completed the present invention.

That is, the present invention provides a method for polymerizing a phenolic compound or the like, a method for producing a compound having an increased molecular weight, an agent (composition) for various applications utilizing the polymerizing method, a method for producing the same, and a method for soil treatment , Porous article processing method, processed material And a method for manufacturing the same.

[1] The following characteristics:

(1) Action: oxidizes polyphenol;

(2) Optimal reaction pH: ^^ Optimum reaction 1 ^ around 8.2 ~ 8.5;

(3) optimum reaction temperature: having an optimum reaction temperature around 50 ° C;

(4) molecular weight: the molecular weight measured by gel filtration chromatography (GFC) analysis is in the range of about 33,000 to 76,000;

(5) Isoelectric point: isoelectric point measured by isoelectric focusing is in the range of about 5.9 to 7.0;

A method for polymerizing a phenol compound, an alkoxyl group-containing aromatic compound, a halogenated phenol compound, a quinone compound, or an aromatic amine compound, which comprises reacting a polyphenol oxidase having the formula:

[2] The method for polymerizing according to [1], wherein the polyphenol oxidase is allowed to act in an alkaline region having a pH of 8 or more.

[3] The method for polymerizing according to the above [1] or [2], wherein polyphenoloxidase is used together with a substance having a peroxidase action.

[4] The method according to any one of [1] to [3], wherein polyoxaloxidase and a substrate thereof are used together with polyphenol oxidase.

[5] The above-mentioned method wherein air, oxygen, ozone, hydrogen peroxide, hydrogen peroxide precursor, peracid precursor or peracid is used alone or in combination as an oxidizing agent.

The method for polymerizing according to any one of [1] to [4].

[6] The following characteristics:

(1) Action: oxidizes polyphenol;

(2) Optimum reaction pH: ρΗ8.2 to 8.5 with an optimum reaction pH;

(4) molecular weight: the molecular weight measured by GFC analysis is in the range of about 33,000 to 76,000; (5) Isoelectric point: isoelectric point measured by isoelectric focusing is in the range of about 5.9 to 7.0;

A compound having an increased molecular weight characterized in that a polyphenol oxidase having the formula (1) is reacted with a phenol compound, an alkoxy group-containing aromatic compound, a halogenated phenol compound, a quinone compound, or an aromatic amine compound to form a polymer, thereby increasing the molecular weight. Manufacturing method.

[7] The following characteristics:

(1) Action: oxidizes polyphenols;

(2) Optimum reaction pH: has an optimum reaction pH around pH 8.2 to 8.5;

(4) molecular weight: the molecular weight measured by GFC analysis is in the range of about 33,000 to 76,000;

A polyphenol oxidase having a phenolic compound, an aromatic compound containing an alkoxy group, a halogenated phenolic compound, a quinone compound, or an aromatic amine compound, an unsaturated fatty acid, an unsaturated alcohol, an unsaturated alkyl compound, or a dry compound. A method for producing a compound having an increased molecular weight, wherein the compound is reacted with an oil to effect a polymerization reaction.

[8] The following characteristics:

(1) Action: oxidizes polyphenol;

(2) Optimum reaction pH:! Has an optimum reaction 1 ^ around 8.2 to 8.5;

A polyphenol oxidase having a phenolic compound, Silyl group-containing aromatic compound, halogenated phenol compound, quinone compound, or aromatic amine compound, and fragrance, deodorant, fire retardant, flame retardant, antibacterial, antiseptic, bactericide, insect repellent A method for producing a compound having an increased molecular weight, which comprises reacting a mixture with at least one drug selected from a drug, an antiviral agent, and a biorepellent to carry out a polymerization reaction.

[9] The method for producing a compound having an increased molecular weight according to [8], wherein the drug is a solution or fine powder of a metal salt, a metal compound, or a metal complex.

[10] The method for producing a compound having an increased molecular weight according to the above [9], wherein the metal is at least one selected from copper, arsenic, zinc, chromium, nickel, aluminum, molybdenum, magnesium and silver.

[11] The method for producing a compound having an increased molecular weight according to the above [8], wherein the drug is a solution or fine powder of a boron salt, a boron compound, or a boron-containing complex.

[12] The method for producing a compound having an increased molecular weight according to the above [8], wherein the drug is a plant-derived extract, an extract component, or a synthetic product having the same structure as the plant extract component.

[13] The production of the compound having an increased molecular weight according to the above [8], wherein the drug is an aromatic compound or a cyclic compound having one or more substituents selected from a hydroxyl group, an amino group, a halogen, and a nitro group. Method.

[14] The method for producing a compound having an increased molecular weight according to any one of the above [6] to [13], wherein the phenolic compound and / or the aromatic amamine compound is lignin or a lignin derivative.

[15] The method for producing a compound having an increased molecular weight according to [14], wherein the lignin derivative is ligninsulfonic acid or a ligninsulfonic acid salt.

[16] The method for producing a compound having an increased molecular weight according to any one of the above [6] to [15], wherein polyphenol oxidase acts in an alkaline region having a pH of 8 or more.

[17] Polyphenoloxida together with a substance having a peroxidase action The method for producing a compound having an increased molecular weight according to any one of the above [6] to [16], wherein the compound is used.

[18] The method for producing a compound having an increased molecular weight according to any one of the above [6] to [17], wherein polyphenol oxidase is used together with oxidase and its substrate.

[19] The above-mentioned [6] to [18], wherein air, oxygen, ozone, hydrogen peroxide, hydrogen peroxide precursor, peracid precursor or peracid is used alone or in combination as an oxidizing agent. The method for producing a compound having an increased molecular weight according to any one of the above.

[20] A thickener, a stabilizer, a flocculant, an emulsifier, and a dispersion, characterized by using the method for producing a compound having an increased molecular weight according to any one of the above [6] to [19]. Agents, water retention agents, humidity control agents, antioxidants, adhesives, dyes, paints, fire retardants, flame retardants, oil recovery agents, soil modifiers, seed spray topsoil stabilizers, deodorants, deodorants Odorant, pesticide spreading agent, oxygen absorber, preservative, bactericide, antibacterial agent, virus infection inhibitor, biofouling inhibitor, biological repellent, insecticide, insect repellent, packing agent, ink base, A method for producing a concrete admixture or a wood treating agent.

[21] A soil treatment method, wherein a reaction for performing the production method according to any one of [6] to [19] is performed in soil.

[22] A method for treating a porous article, wherein the reaction for performing the production method according to any one of [6] to [19] is performed in a porous article.

[23] The method for treating a porous article according to the above [22], wherein the reaction liquid is impregnated into the porous article by pressurization and / or depressurization.

[24] The method for treating a porous article according to the above [23], wherein the pressurization is performed at 1 to 20 atm.

[25] The porous article is a metal sintered body, manufactured article, alloy, die cast article, ceramitas, brick, concrete, wood, wood processing material, fir, rush, straw, bamboo material, or synthetic resin foam Any of the above [22] to [24] A method for treating a porous article according to any of the preceding claims.

[26] A strength, abrasion resistance, weather resistance, weather resistance, flame retardancy, antibacterial property, and antiseptic, characterized by using the treatment method according to any one of the above [22] to [25]. Properties, bactericidal, insect repellent, insecticidal, antiviral, biorepellent, adhesive, sustained release of drug, coloring, dimensional stability, anti-cracking, deodorant, deoxygenating, humidity control, water absorption , Water repellency, surface smoothness, biocompatibility, ion exchange, formaldehyde absorption, drug leaching prevention, or prevention of migration of inorganic compounds to the porous article surface, or improved porosity The method of manufacturing the article.

[27] A thickener, a stabilizer, a flocculant, an emulsifier, an emulsifier, a dispersant, a water retention agent, and a humidity controller containing the compound having an increased molecular weight produced by the method according to any of the above [6] to [19]. Agents, antioxidants, adhesives, dyes, paints, fire retardants, flame retardants, oil recovery agents, soil modifiers, seed spray topsoil stabilizers, deodorants, deodorants, pesticides, spreading agents, Oxygen absorbers, preservatives, bactericides, antibacterial agents, virus infection inhibitors, biofouling inhibitors, biological repellents, insecticides, insect repellents, cataplasms, ink bases, concrete admixtures or wood treatments. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the relationship between the relative pH and the reaction pH of polyphenol oxidase produced by Stillella sp. SD3101 (Stilbella SD3101).

FIG. 2 is a graph showing the relationship between the reaction pH and the relative activity of polyphenol oxidase produced by Sagenomera 'SP. SD3102 (SaRenomella sp. SD3102) SD3102.

FIG. 3 is a graph showing the relationship between the reaction pH and the relative activity of polyphenol oxidase produced by Stachylidium sp. SD3103 (Stachylidium sp. SD3103) SD3103.

Figure 4 shows the temperature and phase of polyphenoloxidase produced by SD3101. It is a graph which shows the relationship with activity.

FIG. 5 is a graph showing the relationship between the temperature and the relative activity of polyphenol oxidase produced by SD3102.

FIG. 6 is a graph showing the relationship between the temperature and the relative activity of polyphenol oxidase produced by SD3103.

FIG. 7 is a graph showing the temperature stability of polyphenol oxidase produced by SD3101.

FIG. 8 is a graph showing the temperature stability of polyphenol oxidase produced by SD3102.

FIG. 9 is a graph showing the temperature stability of polyphenol oxidase produced by SD3103.

FIG. 10 is a graph showing the pH stability (relation between treatment pH and residual activity) of polyphenoloxidase.

FIG. 11 is a graph showing the relationship between the reaction pH and the relative activity of a polyphenol oxidase mixture. Detailed description

Hereinafter, the present invention will be described in detail.

[Production bacteria]

Strains belonging to the genus Stilbella used to obtain the polyphenoloxidase used in the present invention include Stilbella annulate, Stilbella 'Stilbella bulbicola', and Stilbella's Ellis. Stilbella sp. (Stilbella sp.) Stilbella sp. force to be mentioned force ^s preferably Sutirubera 's p. SD 3 1 01 (Stilbella sp. SD3101) (1996 year 1 January to 28 days, Japan, Tsukuba, Ibaraki,巿東1 chome 3 Gosho It has been deposited with the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry as FERM P-15963 and transferred to the international deposit on January 8, 1998 as FERM BP-6219. ) Is used.

In addition, strains belonging to the genus Sagenomella used to obtain the polyphenoloxidase used in the present invention include Sagenomella viride and Sagenomella sp. (Sagenomella sp.) SDS lOS (Sagenomella sp.SD3102) (Ministry of International Trade and Industry, Tsukuba-Higashi 1-3-1-3, Ibaraki, Japan on January 28, 1996) FERM P-15964 was deposited with the National Institute of Bioscience and Biotechnology, and transferred to the international deposit on January 8, 1998 as FERM BP-6220.)

In addition, strains belonging to the genus Stachylidium used to obtain the polyphenoloxidase used in the present invention include strains belonging to the genus Stachylidium 'Stachylidium bicolor', Stachylidium theobromae (Stachylidium theobromae), and Stachylidium 'Slipium sp. ) Is preferred. Stachylidium's p. SD 3103 (Stachylidium sp. SD3103)

(Deposited on January 28, 1996 with FERM P-15965 at the Institute of Biotechnology, Industrial Technology Institute, Ministry of Industry and Industry at 1-3 1-3 Higashi, Tsukuba, Ibaraki, Japan, January 8, 1998 Was transferred to the International Depository as FERM BP-6221.)

Regarding the representative strains producing polyfunoxoxidase used in the present invention, the color and shape of colonies when cultured at 25 ° C for 14 to 60 days on potato kilotot agar medium and malt extract agar medium Morphological observation of conidia, conidia formation structure, etc. was performed. As a result, white and viscous settlements similar to Acremonium are formed, forming conidiophores (cinema), and filaments forming elliptical small conidia with a major axis of 3-5 μm. The bacterium was named Stilbella sp. SD3101 (Stilbella sp. SD31Q1), and also formed a white, viscous colony similar to Acremonium, conidia. Pattern A filamentous fungus that forms chain conidia without forming a bunch (cinema) and forms elliptical to lemon-shaped small conidia with a major axis of 2 to 4 im is used as a seedling of Salagenoma sp. Sagenomella sp.SD3102), and a filamentous fungus that forms a dark brown to black viscous colony and has a rotile spore pattern and a fibrous spore, but does not form a spore locus. It was named SD3103 (Stachylidium sp. SD3103).

[Preparation of enzyme]

The polyphenol oxidase used in the present invention can be obtained by culturing a strain belonging to the genus Stilbella or the genus Sagenomella, belonging to the genus Stachylidium and a mutant thereof, It is also possible to prepare using bacteria. That is, the DNA encoding the polyphenol oxidase, together with an appropriate promoter, an operator, and a terminator DNA having an enzyme expression function in the host organism, together with a DNA, serves as an origin of replication for replicating the vector in the host organism. A host cell transformed with an expression vector inserted into a DNA vector having the same, or a suitable promoter having an enzyme expression function in a host organism, wherein the DNA encoding the polinooloxidase has a function of expressing an enzyme in a host organism; Host cells transformed by integration into host cell DNA together with DNA are cultured under conditions that allow polyphenol oxidase to be expressed, and polyphenol oxidase is also recovered from the culture medium. You.

Further, the polyphenol oxidase of the present invention, based on the knowledge about the amino acid sequence of the polyphenol oxidase obtained based on the DNA sequence encoding the polyphenol oxidase, based on the conventional acidic side It may be produced by a protein engineering technique for modifying the DNA of polyphenol oxidase having an optimum reaction pH.

In order to obtain a DNA fragment encoding the polyphenol oxidase of the present invention, for example, cDNA or a genomic library from the strain of the present invention is separated. The ability to identify a DNA fragment of interest using, as a source, an oligonucleotide synthesized based on the amino acid sequence of the polyphenoloxidase of the present invention or the amino acid sequence of a known polyphenoloxidase, or an enzyme. The method can be carried out by a conventional method, such as selecting a clone that expresses a catalytic activity, or selecting a clone that produces a protein that reacts with the antibody against the polyphenoloxidase.

For the culture for obtaining the polyphenol oxidase used in the present invention, a commonly used synthetic medium or a nutrient medium containing an organic carbon source and an organic nitrogen source can be used. Also, C u ² + O.OOlmM from 1 Omm ion as the metal salt, preferred properly is preferably added at a concentration of 1 mM from 0. OlmM. The culture temperature is 10 to 60 ° C, preferably 20 to 40 ° C. An appropriate culture time is 20 hours to 250 hours, preferably 50 hours to 150 hours. The secreted polyphenol oxidase can be recovered from the medium by a known method. This recovery procedure includes a series of procedures in which cells are separated from the medium by centrifugation, filtration, or membrane separation, and chromatography is performed, for example, by ion exchange chromatography. Also, membrane concentration using an ultrafiltration membrane is effective.

[Properties of enzymes]

The polyphenol oxidase used in the present invention has the following properties. (1) Action: oxidizes polyphenol;

(2) Optimum reaction pH: has an optimum reaction pH around ρΗ8.2-8.5;

(5) Isoelectric point: The isoelectric point measured by isoelectric focusing is in the range of about 5.9 to 7.0.

Representative polyphenol oxidases used in the present invention include the following. ① Polyphenol oxide derived from Stilbella sp. (Stilbella sp.) SD3101 (accession number FERM BP-6219):

(1) Action: oxidizes polyphenol;

(2) Optimum reaction pH: has an optimum reaction pH around pH 8.2;

(4) molecular weight: the molecular weight measured by GFC analysis is about 76,000;

(5) Isoelectric point: The isoelectric point measured by isoelectric focusing is about 6.6.

(2) Polyphenoloxidase derived from Sagenomella sp. (Sagenomella sp.) SD3102 (Accession number FERM BP-6220):

(1) Action: oxidizes polyphenol;

(2) Optimum reaction pH: has an optimum reaction pH around ρΗ8.5 ·

(4) molecular weight: the molecular weight measured by GFC analysis is about 33,000;

(5) Isoelectric point: The isoelectric point measured by isoelectric focusing is about 5.9.

(3) Polyphenoloxidase derived from Stachylidium sp. (Stachylidium sp.) SD3103 (Accession No. FERM BP-6221):

(1) action: oxidizes polyphenol;

(2) Optimum reaction pH: Optimum reaction around ^ 18.2? Having 1;

(4) molecular weight: the molecular weight measured by GFC analysis is about 55,000;

(5) Isoelectric point: The isoelectric point measured by isoelectric focusing is about 7.0.

These three types of polyphenol oxidases can perform the oxidation reaction in a wide pH range of 5 to 11, but are preferably pH 6 to 10, more preferably pH 7 to 9, and have an optimum pH around pH 8, It has the advantage of catalyzing the oxidation reaction from alkaline to alkaline (Figs. 1 to 3). In addition, the optimal temperature for a 10-minute reaction at pH 8 is around 50 ° C (Figs. 4 to 6), and furthermore, after a 30-minute heat treatment at various temperatures. The activity showed approximately 100% residual activity in the range of 50 ° C. or less (FIGS. 7 to 9). Furthermore, various The remaining activity after treatment at 30 ° C. for 30 minutes in a buffer at pH was stable over a wide range of pH (FIG. 10). These results guarantee oxidation in a wide range of pH from neutral to alkaline in a variety of medium and low temperature solutions.

It is also possible to use the polyphenol oxidase according to the present invention in combination with a conventional enzyme having an optimal reaction pH on the acidic side. In other words, by using a conventionally known polyphenol oxidase having an optimum reaction pH on the acidic side in combination with the polyphenol oxidase of the present invention, a wide pH range from acidic to alkaline can be obtained. , A polyphenol oxidase reaction can be performed. When a mixture of enzymes is used for such a purpose, the mixing ratio of the activity of the polyphenol oxidase having the optimal reaction pH on the acidic side to the activity of the polyphenol oxidase according to the present invention is preferably 1:10 to 10: 1, more preferably 1: 3 to 3: 1. Thus, the polyphenol oxidase according to the present invention is also useful for achieving a polyphenol oxidase reaction in a wide pH range.

[Activity measurement method]

In the present invention, the activity of the polyphenol oxidation activity was measured at 25 ° C in an aqueous solution containing 20 ppm syringaldazine and 100 mM potassium phosphate buffer solution (ρΗ8.2). This was performed by measuring the absorbance at 525 nm. The amount of activity that oxidizes 1 // mo1 of syringal dazine per minute was defined as 1 unit (Unit; hereinafter, abbreviated as U).

[Phenol compound, aromatic compound containing alkoxyl group, halogenated phenol compound, quinone compound and aromatic amine compound]

The phenolic compound, alkoxyl group-containing aromatic compound, halogenated phenol compound, quinone compound, or aromatic amine compound to be polymerized in the present invention is any compound as long as the enzyme used in the present invention can be oxidized. Can also be used. Specific examples of such compounds include lignin, ligninsulfonic acid, humic acid, nitrohumic acid, tannin, catechin, Gallic acid, urushiol, hesperidin, chlorogenic acid, hinokitiol, pyrocatechol, hydroquinone, O-coumaric acid, p-coumaric acid, coniferyl alcohol, coniferyl aldehyde, 3,4-dihydroxybenzoic acid, tert-butyl high Droquinone, phenylhydridoquinone, trimethinole hydroquinone, 3,4-dihydroxycinnamic acid, ethyl-3,4-dihydroxycinnamic acid, pyrogallol, 4-hydroxydroxycinnaminoleanolecone, la perylgallate, octylgallate, syringic acid Sinapyr alcohol, Sinapic acid, Sinapine aldehyde, Homovanilla acid, Homovanillyl alcohol, Homovanillinyl nitrile, Phenoleric acid, Vanillin, o—Vaniline, Vanilla acid, Nonanilyl alcohol, Vanillylamine, Vanillylazine, Asconolevinic acid, 1,2-Dihydroxynaphthalene, 2,3-Dihydroxynaphthalene, 6,7-Dihydroxy-2-naphthalenesulfonic acid, Anthralobin, Alizarin, Quinizarin, o-Feni Rangenamine, p-phenylenediamine, 3,4-diaminobenzophenone, o-anisidine, p-anisidine, o-aminophenol, p-aminophenol, 1,2-diaminoanthraquinone, 1,4-diene Examples include compounds such as aminoaminosuraquinone and compounds obtained by derivatizing these compounds.

In addition to these compounds, any substance that can oxidize the enzyme used in the present invention can be used as a raw material of a polymerized substance or as a catalyst for a polymerization reaction. Examples of such compounds are 2,2 '.- azobis (3-ethylbenzothiazoline-16-sulphonic acid) (ABTS), pyrirubin, isoascorbic acid, quercetin, rutin, guaiacol, 4-methoxyphenol, biphenol , 4, A'-Ethylenedianiline, Methylhydroquinone, Ethylhydroquinone, o-Hydroxybenzoic acid, p-Hydroxybenzoic acid, 1-Hydroxybenzotriazole, 6-Hydroxy-1,2,4, 5—Triaminopyrimidine, 4, 5, 6—Triaminopyrimidine, 2,3-Dihydroxypyridazine, 3, 6—Dihydroxypyridazine, 2,3-Dihydroxypyridin, 4-Hydroxy-3-methoxybenzoic Acid, methyl 4-hydroxy-1 3 Methoxybenzoic acid, 4,5-diamino 6-hydroxy-1-2-mercaptopyrimidine, 2,3-diaminopyridine, 2,5-dihydroxy-11,4-benzoquinone, 2,5-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,4-dihydroxy-3-cyclobutene-1,2-dione, 3- (3,4-dihydroxyphenyl) 1-L-alanine, 2-amino-3-hydroxypyridine, 3- Amino-2-methoxydibenzofuran, 2,4-dimethoxyaniline, 2,5-dimethoxyaniline, 3,4 dimethoxyaniline, 2 ', 5'-dimethoxyacetophenone, 3', 4'-dimethoxyacetophenone , 1,4-Dimethoxybenzene, Veratrol, 2,3-Dimethoxybenzoic acid, 2,5-Dimethoxybenzoic acid, Veratric acid, Veratrua Dehydro, vera torinoleamine, homobate tolonic acid, homobate triluamine, homobate mouth acetate, 3,4 dimethoxybenzinole alcohol, 3,4 dimethoxy cinnamate, 3,4-dimethoxy Cinnamonitrile, 2,3-Dimethoxyphenol, 3,4-Dimethoxyphenol, 3,4-Dimethoxyphenethylamine, (3,4-Dimethoxyphenyl) acetic acid, 3,4-Dimethoxystyrene , (3,4-Dimethoxyphenyl) acetonitrile, 4-aryl-1 2-methoxyphenyl, 2-methoxy41-profeninophenol, 2-methoxy-1-5-methylaniline, 2-methoxy 1-5-Nitroaniline, 4-methoxy 2-Nitroaniline, 3-methoxysalicylic acid, 3-methylcatechol, 4-methylcatechol, methylgas 3,4-, 5-trimethoxyaniline, 3,4,5-trimethoxyphenol, trobolone, purpurogallin, salicylaldoxime, 3-amino-5,6,7,8-tetrahydro-1- Naphthol, 1,5-dihydroxynaphthalene, 3,5-dihydroxy-1-naphthoic acid, 4-hydroxy-1-naphthalene-nolephonic acid, purpurin, 2,3-dihydro-1--9,10-dihi Droxy-1,4-anthracenedione, various azo dyes, and derivatives of these compounds. In addition, a plurality of these compounds can be used in combination for the purpose of adjusting the physical properties of the polymerized product. Further, when producing a polymer compound according to the present invention, a quinone compound which is polymerized by a similar reaction route may be allowed to coexist. Examples of such quinone compounds include anthraquinone 1-2-sulfonic acid, anthraquinone 1-1,5-disnoroleic acid, anthraquinone 1-2,6-disulfonic acid, anthraquinone-2-carboxylic acid, 1-aminoanthra Quinone, 2-aminoaminosuraquinone, anthralphine, aminonaphthoquinone, 1,8-dihydroxylaquinone, camphoquinone, dehydroascorbic acid, 2-hydroxy-1,4-naphthoquinone, isatin, 5-nitroisatin And various anthraquinone dyes. Also, unsaturated fatty acids such as oleic acid, linoleic acid, and linolenic acid, or unsaturated alcohols such as oleyl alcohol, or unsaturated alkyls such as squalene, and dryness such as tung oil, linseed oil, and castor oil Oxidation and polymerization can be performed simultaneously with the enzymatic reaction by coexisting a substance to be auto-oxidized such as oil.

[Polymerization reaction method and its use]

Applications of the novel polyphenol oxidase having an optimum reaction pH in the alkaline pH range include, for example, fuynol compounds, alkoxyl-containing aromatic compounds, halogenated phenol compounds, quinone compounds, and aromatic amines. There is an application to a polymerization reaction of a compound. In the production of the phenolic compound, the alkoxyl group-containing aromatic compound, the halogenated phenolic compound, the quinone compound, or the aromatic amine compound having an increased molecular weight by the polymerization reaction of the present invention, the concentration of these compounds is 0.01 to 0.1%. It is 90% by weight, preferably 1 to 80% by weight. The reaction temperature is 0 to 100 ° C, preferably 0 to 70 ° C. In addition, the pH of the reaction is 5-11, preferably 6-10, more preferably 7-9.

The enzyme activity concentration used is 1 to 10,000 UZ liter, preferably 10 to 2000 U / L. It is desirable to adjust the enzyme activity concentration according to the purpose. That is, when it is desired to achieve rapid polymerization and gelation or solidification, the reaction may be performed at a high active concentration. On the other hand, the reaction was performed at a low activity concentration. For example, a moderate polymerization reaction proceeds, and a more uniform solution of the polymerization product can be obtained as a liquid substance. When the reaction is further continued, a gentle gelation reaction proceeds throughout the reaction solution. Stopping the reaction at ItaruTsuta time to a suitable degree of polymerization, Na OH, NH _3, Na ₂ C_〇 _3, added force of an alkali or alkali salts, such as C a CO _3! ], Addition of acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, carbonic acid, boric acid, organic acids, addition of known enzyme inhibitors, heat treatment at 100 ° C for 15 minutes, or supply of oxygen Can be implemented by shutting off. The gelled phenolic compound, aromatic compound containing an alkoxyl group, halogenated phenolic compound, quinone compound, or aromatic amine compound may be dissolved again by heating at 50 to 230 ° C if desired. Is possible. Such thermal solubility is a useful property when used in applications such as dispersants, adhesives, and paints. Also, by adding hot water or the like after heat dissolution and dispersing and dissolving, it is possible to obtain a compound having a very high molecular weight as a solution.

Further, according to the method of the present invention, metal sintered bodies, manufactured articles, alloys, die cast articles, ceramics, bricks, concrete, wood, woodwork, fir, rush, straw, bamboo, synthetic resin foam, etc. In the treatment of porous articles, polyphenol oxidase is impregnated with both a phenol compound, an aromatic compound containing an alkoxyl group, a halogenated phenol compound, a quinone compound, or an aromatic amine compound, and a polymerization reaction is performed in the porous article. In this case, it is desirable that the treatment liquid before the impregnation does not gel or solidify, and the gelation and solidification proceed when the treatment liquid is dried and concentrated in the porous article after the impregnation. For this purpose, the concentration of the phenol compound, the alkoxyl group-containing aromatic compound, the halogenated phenol compound, the quinone compound or the aromatic amine compound in the treatment solution is 0.01 to 50% by weight. /. Preferably, it is 0.1 to 30% by weight. The reaction temperature is 0 to 100 ° C, preferably 10 to 70 ° C. Furthermore, the pH of the reaction is between 5 and 11, preferably between 6 and 10, more preferably between 7 and 9. In addition, the enzyme activity concentration to be used is:! To 10,000 U / liter, preferably 1 O to 2000 U / liter. Termination of the reaction at the time that led to a suitable degree of _{polymerization, N a OH, NH 3,} Na 2 C_〇 _3, Impregnation of alkali or alkali salts, such as C a CO _3, hydrochloric acid, sulfuric acid, impregnation of an acid such as nitric, impregnation of known enzyme inhibitors, heat treatment such as 1 0 0 ° C, 1 5 minutes, Oh Rui porous It can be implemented by coating the surface of the product with oxygen and shutting off the oxygen supply by packaging with film.

In order to further increase the effect of the enzymatic polymerization reaction, polyphenol oxidase and a phenol compound, an aromatic compound containing an alkoxyl group, a halogenated phenol compound, a quinone compound, or an aromatic amine compound, and an unsaturated compound Fatty acids, unsaturated alcohols, unsaturated alkyl compounds, and unsaturated compounds such as drying oils are polymerized together to form phenolic compounds by enzymatic or automatic oxidation, aromatic compounds containing alkoxyl groups, halogenated phenols The polymerization reaction of the compound, the quinone compound, or the aromatic amine compound and the polymerization reaction based on the autoxidation of the unsaturated compound are simultaneously performed, so that a stronger composite can be obtained. The concentration of the unsaturated compound used for such purpose in the treatment solution is 0.001 to 60% by weight, preferably 0.01 to 40% by weight. According to the present invention, polyphenol oxidase is impregnated into a porous article by pressurizing and / or depressurizing, and is already contained in a porous article, such as a polyphenol compound such as lignin already contained in wood. For example, when the porous article is wood, the workability in the drying step after the wood impregnation treatment can be improved by polymerizing the polyphenol compound, aromatic amine compound, etc. Improved wood strength reduced by lignin decomposition by wood steaming treatment or high-temperature steam injection, improved action to prevent cracking of wood during drying or freezing, maintenance of anaerobic environment in wood, and suppression of microbial growth by improvement Becomes possible. In order to further enhance the effect of the enzymatic polymerization reaction in the porous article, polyolefin oxidase and a phenol compound, an aromatic compound containing an alkoxyl group, a halogenated phenol compound, a quinone compound, or an aromatic amine are used. By impregnating the porous article together with the pressurized compound and / or depressurizing it and conducting a polymerization reaction in the porous article, the product does not contain or contains a substance on which polyphenoloxidase acts. Effective treatment of porous articles with less In addition, since the enzymatic polymerization reaction mainly proceeds inside the porous article, a treatment liquid having a relatively low molecular weight of the reaction solution composition, that is, a treatment liquid having a relatively low viscosity is used. Thus, a large amount of the processing solution can be easily impregnated. In particular, when a substance such as lignin, on which polyphenoloxidase can act, already exists and is immobilized in the porous article, this is caused by the enzymatic action in the porous article, resulting in a fuanol compound or an aromatic compound containing an alkoxyl group. Reacts with an aromatic compound, a halogenated phenolic compound, a quinone compound or an aromatic amine compound, and polymerizes, resulting in a phenolic compound, an alkoxyl group-containing aromatic compound, a halogenated phenolic compound, a quinone compound or an aromatic amine compound. The resulting polymer is more firmly immobilized in the porous article.

The pressurization or depressurization performed for the purpose of the present invention is extremely important in injecting a sufficient amount of the processing liquid into various types of porous articles that are difficult to impregnate with the processing liquid, and to obtain a necessary processing effect. is there. The pressurization operation is performed at atmospheric pressure (1 atm) to 20 atm, more preferably at 3 to 15 atm, but if the enzyme activity is not lost, higher pressure is applied. It is also possible. In addition, the decompression operation can be performed at any pressure up to the vacuum pressure.However, for effective treatment of porous materials that are difficult to impregnate, a pressure in the range of 100 to 76 OmmHg is required. It is desirable to reduce the pressure. In addition, it is more preferable that the depressurizing operation be performed by evacuation before depressurizing before adding the processing liquid to the porous article. In order to impregnate the treatment liquid in a larger amount in the porous article, it is also effective to carry out a combination of these pressure operation and pressure operation.

When the porous article is wood, various commonly used pressurizing and depressurizing methods can be used, specifically, a packed cell method (Vesel method), a semi-empty cell method (Lowry method), and a double vacuum method. The method (double vacuum method), the pressure change method (Oscillating Pressure Method), and the method combining these operations are applicable. Also, the insizing method can be applied to increase the impregnation amount. In addition, as a pre-treatment of porous articles that are difficult to impregnate, compression treatment using rollers, microwave heating, freezing treatment, steaming treatment, steam treatment, or heat treatment It is also effective to perform processing. Originally, it is known that lignin is contained abundantly in wood core material, thereby increasing the resistance to rot fungi and termites. Therefore, when the phenol compound, the alkoxyl group-containing aromatic compound, the halogenated phenol compound, the quinone compound, or the aromatic amine compound of the present invention is lignin or a lignin derivative, and is used as a wood preservative, INDUSTRIAL APPLICABILITY The treatment method of the present invention enables a method for protecting against rot fungi and termites, which are essentially carried out on natural living wood, to be efficiently implemented as an industrial treatment method for all types of wood. .

Lignin, or ligninsulfonic acid or ligninsulfonate, contains various water-insoluble solid components as produced from the process of cooking pulp or sulfite pulp. Therefore, when the porous article is impregnated with polyphenoloxidase and lignin or ligninsulfonic acid or ligninsulfonate, and the polymerization reaction is performed in the porous article, the porous article is used for the purpose of the present invention. In order to increase the amount of the treatment liquid impregnated in the quality article, it is desirable to remove the water-insoluble solid components in these pulp waste liquids. The removal treatment is performed by a method such as centrifugation, filtration, and standing according to the type of the porous article to be impregnated, the purpose of the impregnation treatment, and the economics involved in the removal. For example, for the purpose of performing pressure injection treatment on wood, the diameter or major diameter in the waste pulp 以上 μππι or more, preferably 0.5 m or more, more preferably 0.1 // m or more It is desirable to remove the water-insoluble solid components by filtration. It is also possible to use those that have been desalted and desugared by ultrafiltration, or fractions having a lower molecular weight such as a molecular weight of 0.5000 to 100,000 or less in order to further enhance impregnation. Desaccharification can also be performed by a microorganism such as yeast. The lignin derivative used for the purpose of the present invention may be lignin or lignin sulfonic acid, in addition to lignin sulfonic acid, acetic esterification, propionate esterification, carboxymethyl etherification, 2-hydroxylesteryl esterification, 2-acetoxityl etherified or 2-hydroxypropyl etherified, and lignin or lignin derivatives thereof Can be used. Also, mixtures thereof can be used.

Among the phenol compounds, alkoxyl group-containing aromatic compounds, halogenated phenol compounds, quinone compounds and aromatic amine compounds used in the present invention, particularly, lignin, ligninsulfonic acid, humic acid, nitrohumic acid, tannin, catechin, Natural products or natural product derivatives such as gallic acid, urushiol, hesperidin, and hinokitiol are highly useful because of their high safety to the environment and the human body.

It is also effective to apply or impregnate a drug to the porous article as a pre-treatment or post-treatment of the polymerization reaction treatment in the porous article. In particular, the first step of applying or impregnating the drug on the porous article for the purpose of sealing the drug inside the porous article, and in particular, preventing the migration of the inorganic compound to the surface of the porous article, and polyphenol. Impregnating a porous article with oxidase and both a phenolic compound, an aromatic compound containing an alkoxyl group, a halogenated funinol compound, a quinone compound, or an aromatic amine compound under pressure and Z or under reduced pressure A porous article treatment method performed in two steps is useful. Conversely, both polyphenoloxidase and a phenolic compound, an alkoxyl group-containing aromatic compound, a halogenated phenolic compound, a quinone compound, or an aromatic amine compound are pressurized and / or depressurized to form a porous article. The first stage of impregnation and the use of the interaction of the drug with a phenolic compound, an aromatic compound containing an alkoxyl group, a fluorinated halogenated compound, a quinone compound, or an aromatic amine compound to put the drug inside the porous article For the purpose of fixing, a porous article treatment method that is performed through a second step of applying or impregnating a porous article with a drug is also useful for effective drug treatment. The present invention in combination with pre-treatment or post-treatment as compared to a case where a porous article is simply impregnated with a phenol compound, an alkoxyl group-containing aromatic compound, a halogenated phenol compound, a quinone compound, or an aromatic amine compound. According to the method, a phenol compound, an alkoxy group-containing aromatic compound, a halogenated phenol compound, a quinone compound, Since the aromatic amine compound is polymerized and fixed in the porous article by the enzyme-catalyzed reaction, the drug can be more firmly fixed.

In addition, after the treatment liquid is impregnated into the porous article, a method of performing a decompression treatment to recover a part of the treatment liquid outside the porous article, and a sufficient polymerization reaction inside the treated porous article. Before proceeding, the porous article is washed with water or the like to remove unpolymerized substances, so that the degree of maintaining the porous property can be easily adjusted. The treated material that has maintained and adjusted such porosity retains the ability to adjust humidity, water retention, adsorption, and ion exchange, and can be used in a variety of applications that utilize these capabilities. is there. Further, a porous article having various composite properties can be manufactured by further impregnating the treated material with the retained porous property with a drug, a polymer, or a prepolymer.

The purpose of increasing the effect of the porous article treatment of the present invention, and the ability to immobilize a drug by a phenol compound, an alkoxyl group-containing aromatic compound, a halogenated phenol compound, a quinone compound, or an aromatic amine compound polymerized inside the porous article. And various fragrances, deodorants, antibacterial agents, flame retardants, antibacterial agents, preservatives, bactericides, insect repellents, antiviral agents, biological repellents Can be performed as a pre-treatment, a simultaneous treatment, or a post-treatment of the porous material treatment with polyphenoloxidase. Many existing drugs can be used for this purpose. Usable chemicals include not only water-soluble chemicals, but also those that form OZW-type or W / O-type emulsions by the addition of dispersants or surfactants, or as fine powders in aqueous solutions. Dispersing ones are also available.

Surfactants used for such purposes include, for example, linear or branched alkyl or alkenyl sulfates, amide sulfates, linear or branched alkyl or alkenyl groups, ethylene oxide, Aliphatic sulfates such as alkyl or alkenyl ether sulfates to which one or more of propylene oxide and butylene oxide have been added, alkyl sulfonates, amide sulfonates, dialkyl sulfosuccinates, phosphorates Aliphatic sulfonates such as fin, vinylidene type olefin and 內 part olefin sulfonate, aromatic sulfonates such as linear or branched alkylbenzene sulfonic acid, linear or branched alkyl group or Alkyl or alkenyl ether carboxylate or amide having a alkenyl group, to which one or more of ethylene oxide, propylene oxide and butylene oxide are added, a sulfo fatty acid salt or an ester. , Amino acid type surfactants, alkyl or alkenyl acid phosphates, phosphate ester surfactants such as alkyl or alkenyl phosphates, sulfonic acid amphoteric surfactants, betaine amphoteric surfactants, linear or branched A chain alkyl or alkenyl group Having an alkyl or alkenyl ether or alcohol to which one or more of ethylene oxide, propylene oxide and butylene oxide are added, a linear or branched alkyl or alkenyl group, and ethylene oxide Polyoxyethylene alkyl phenyl ether to which one or more of propylene oxide and butylene oxide are added, higher fatty acid alkanolamide or its alkylene oxide adduct, sucrose fatty acid ester, fatty acid glycerin monoester, Examples thereof include an alkyl or alkenyl amine oxide, a tetraalkyl ammonium salt type cationic surfactant, and the like. Many conventionally known dispersants can be used.Lignin, ligninsulfonic acid, or ligninsulfonate is particularly a raw material for the polymerization reaction of polyphenoloxidase. However, since these substances themselves have a drug dispersing action, they are useful for the purpose of the present invention. Copper, arsenic, zinc, chromium, nickel, aluminum, molybdenum, magnesium as the above agents used for the purpose of the present invention, as antibacterial agents, preservatives, bactericides, insect repellents, antiviral agents, or biological repellents Alternatively, a solution or fine powder of a silver metal salt, a metal compound, or a metal complex can be used. Specifically, the anion moiety is F -, CI -, B r -, N 0 3, BO -, P_〇 ³ -, S 2 -, metal salts consisting of C CK ^2, etc., naphthenic acid, Orein acid And the like, a compound of a carboxylic acid or a sulfamic acid and a metal ion, a metal oxide, a metal oxide ion, or a complex thereof. In addition, calcium bromide, sodium bromide, magnesium bromide, potassium bromide, sodium iodide, sodium fluoride, potassium fluoride, sodium potassium fluoride, magnesium fluoride, sodium sulfide, potassium sulfide, selenium Acid reames can also be used.

Many conventionally known compounds can be used as compounds added to form a complex with a metal. For example, phenolic compounds such as pyrocatechol, gallic acid, hinokitionore, catechin, pyrogallol, o-phenylenediamine, 2-aminophenol or aromatic amine compounds, ethane-1,1-diphosphonic acid and derivatives thereof, ethanehydroxyl Phosphonic acids such as 1,1,2-triphosphonic acid, 1,2-dicanoleboxyl-1,2-diphosphonic acid and methanehydroxyphosphonic acid, 2-phosphonobutane 1,2-dicanolevonic acid, and 1-phosphonobutane 12 2,3,4-Tricarboxylic acid, phosphonocarboxylic acid such as ct-methylphosphonosuccinic acid, amino acid such as aspartic acid, glutamic acid, glycine, 2-aminoisobutyric acid or amino acid analog, tri-triacetic acid, ethylenediamine Tetraacetic acid, diethylenetriaminepentaacetic acid Polyamino acids such as aminopolyacetic acid, polyacrylic acid, polyitaconic acid, polymaleic acid, maleic anhydride copolymer, carboxymethinoresenorelose, etc., non-dissociable polymers such as polyethylene glycol and polyvinyl alcohol, diglycolic acid, Carboxymethylated products such as oxydisuccinic acid, carboxymethyloxysuccinic acid, dalconic acid, citric acid, lactic acid, tartaric acid, sucrose, lactose, carboxymethylated pentaerythritol, carboxymethylated gluconic acid, benzenepolycarboxylic acid Organic acids such as oxalic acid, linoleic acid, oxydisuccinic acid, dalconic acid, ethylenediamine, ethanolamine, diethanolamine, triethanolamine, triethylenetetramine, polyethyleneimine Organic alkali agents such as triisopropanolamine § Min, starch, urea, chitosan, organic materials such as polylysine No.

The above hinokitiol can be used as various metal complexes or salts. Specific examples include complexes with copper, arsenic, zinc, chromium, nickel, aluminum, molybdenum, magnesium, calcium, barium, iron or silver, or sodium salts. In particular, a complex with copper, arsenic, zinc, chromium, nickel or silver can be used for the purpose of the present invention since a metal can be added to the physiological action of hinokitiol, such as antibacterial action. . As the metal powder, a powder composed of metal fine particles of various sizes can be used depending on the purpose. Hereinafter, more preferably, a powder composed of fine particles having a diameter of 0.1 m or less can be used.

The treatment of the porous article with a metal salt, a metal compound, or a metal complex can be performed by any of the pretreatment, post-treatment, and simultaneous treatment of the porous article with polyphenoloxidase. Depending on the degree to which the polymerization reaction is inhibited, the solubility under the enzyme reaction conditions, the presence or absence of aggregation and sedimentation when mixed with the treating agent, the purpose of the treatment, etc., in various methods or in various combinations. Can be. The concentration of the metal salt, metal compound, or metal complex in the treatment solution is preferably adjusted according to the strength of the physiological activity of the metal used and the purpose of the treatment.For example, copper, arsenic acid, or zinc In this case, it is usually 0.01 to 50 OmM, preferably 0.1 to 20 OmM. Also, solutions or fine powders of boron salts, boron compounds, or boron-containing complexes can be used as flame retardants, antibacterial agents, preservatives, bactericides, insect repellents, antiviral agents, or biological repellents. Specific examples include boric acid, borax, and copper borofluoride. Further, pyrethroid drugs, conazole drugs, insect hormone drugs and the like can also be used as the drug of the present invention. By imparting and improving the sustained-release property of these volatile drugs to the porous article according to the present invention, the efficacy can be maintained for a longer period.

Also, fragrance, deodorant, antibacterial, antibacterial, antiseptic, bactericide, insect repellent, antibacterial As a virus agent or a biological repellent, a plant-derived extract, an extract component, or a compound having a structure equivalent to that of a plant extract component can be used. Specific examples of such plants include trees such as hinoki and Aomori hiba, herbs, radish, rust, bamboo, iriomote thistle rhizome, or yaeyama palm root.These plants are crushed, squeezed, Extracts and extractable components can be obtained by boiling or steam distillation. Specific examples of plant-derived components or compounds having a structure equivalent to that of plant extracts include tropolones such as hinoki chionore, hyibinene,] 3-binene, camphor, menthol nore, limonene, Bonneol, α-tenorebinene, γ-terbinene, mono-tenore pines such as pineo-pinole, tenorepinene 4-monoole, mono-enoles such as sineo-no-ole, sesquiterpenes such as α-casinole and t-murol, catechin, tanji Such as polyphenols such as butane, naphthalene derivatives such as 2,3,5-trimethylnaphthalene, long-chain aliphatic alcohols such as citronellol, aldehydes such as cinnamaldehyde, citral, perillaaldehyde, and arylisothiocyanate. Aryl compounds and the like. Wood vinegar obtained by steaming trees can also be used.

Since these extracts, extracted components, or composites having a structure equivalent to that of plant extracts are originally present inside the plant, the porous article is made of wood, wood-processed material, wood chips, wood flour. , Fir, rush, straw, bamboo, fiber, paper, or pulp, and the raw material for the polymerization reaction on which polyphenoloxidase acts is lignin or lignin derivatives. In the case of a plant-derived component, in addition to the action of sealing the voids in the porous article by the polymer, a plant-derived extract, an extract component, or a compound having an equivalent structure, and a porous article or a reaction raw material thereof By interacting with, it is possible to obtain desirable effects such as leaching resistance and sustained release of the drug. In addition, the processed products of porous articles produced by combining these natural products have high safety to the environment and the human body, and are biocompatible, so they can be used in various fields. It is. It also has one or more substituents selected from hydroxyl, amino, halogen, and nitro groups as antibacterial agents, antibacterial agents, preservatives, bactericides, insect repellents, antiviral agents, or biological repellents. Aromatic compounds or cyclic compounds can be used. These aromatic compounds are also desirable in terms of the leaching resistance of the drug and the sustained release, based on the same principle as in the case of the above-mentioned plant-derived extract, extract component, or compound having a structure equivalent to the plant extract component. The effect can be obtained.

Specific examples of aromatic compounds or cyclic compounds having one or more substituents selected from a hydroxyl group, an amino group, a halogen, and a nitro group include o-phenylphenol, 1-naphthol, and 2-naphthol. , O-black phenol, 2,4-dinitrophenol, 4,6-dinitro-o-crezo, pentachlorophenol, 2,3,5-trichlorophenol, 2,4,6-tri Black mouth phenol, monochloronaphthalene, trichloronaphthalene, tetrachloronaphthalene, 2, 4, 5 _ Triclo mouth phenolinolelaurate monochrome mouth naphthalene, chloronitrophenol, chloronitrotonolene, o-dichlorobenzene, 1, 3 , 5—Trichloro mouth benzene, 1, 2, 4—Trichloro mouth benzene, 2, 4, 6—Tribromophenolone 4-Bromo-2,5-dichlorophenol phenol, bromo-o-phenolenoate, 4-chlorofenolene 3-propadylformal, creosote oil, chlorinated terpene, butylhydroxyanisole, butylhydride Examples include roxytoluene, benzoic acid, p-hydroxybenzoic acid, and esters of p-hydroxybenzoic acid such as methyl, ethyl, propyl, butynole, isobutyl, and isopropyl. Also, organophosphorus compounds such as thiofamate methyl, dasban, diazinon, cyclogen compounds such as chlordane, dieldrin, aldrin, heptachlor, carbamate compounds such as bigon, dimethylene, sevin, etc., and N-nitroso-N-cyclo Nitroso compounds such as xylhydroxylamine, dehydroacetic acid, and sonolevic acid can also be used for the purpose of the present invention as antibacterial agents, preservatives, bactericides, insect repellents, antiviral agents, or biological repellents. The dimensional stability, crack prevention, humidity control, water absorbency, water repellency, and surface smoothness imparted or improved by the porous article treatment of the present invention are based on the hydrophilicity or hydrophobicity of the polymer generated in the porous article. This is achieved by adjusting For example, a polymerized product of lignin sulfonic acid is polymerized into a hydrophilic polymer gel in a porous article, but other treating agent components such as unsaturated fatty acids, unsaturated alcohols, unsaturated alkyl compounds, Hydrophobicity can be improved by using an unsaturated compound such as oil in combination. In addition, an aromatic compound having a saturated or unsaturated alkyl side chain having 1 to 22 carbon atoms as a substituent in addition to a hydroxyl group, specifically urushiol, is a main component to be polymerized by polyphenoloxidase. Hydrophobicity can also be improved by using it or adding it to lignin or lignin derivatives. Further, in addition to a hydroxyl group and / or an amino group, a phenol compound having a substituent having polyoxyethylene or polyethyleneimine as a structural part, an aromatic compound containing an alkoxyl group, a halogenated phenol compound, a quinone compound, or an aromatic compound By using an aromatic amine compound as a main component to be polymerized by polyphenoloxidase, or by adding it to a lignin / lignin derivative, the water retention of the porous article after the treatment can be improved. In wood treatment, dimensional stability and crack prevention can be imparted or improved. In particular, when a phenol compound such as lignin or a lignin derivative, an aromatic compound containing an alkoxyl group, a halogenated phenol compound, a quinone compound, or an aromatic amine compound is used as a main component to be polymerized by polyphenoloxidase, Addition of oxyethylene, polyethyleneimine, or an aromatic compound having a substituent having a saturated or unsaturated alkyl chain having 1 to 22 carbon atoms as a structural part to modify the physical properties of the polymer Can be used as an agent. Aromatic compounds having a substituent having polyoxyethylene or polyethyleneimine as a structural part include ethylene oxide in aromatic compounds having a hydroxyl, amino, carboxyl or the like as a structural part. Can be obtained. Also, Vaniri O-vanillin, 3,4-dihydroxybenzanolaldehyde, benzanolaldehyde, 2-phenylpropionaldehyde, or other aldehyde groups on the aromatic ring or on the aromatic ring It can also be obtained by allowing polyethyleneimine to act on an aromatic compound having a part of the structure of the substituent to generate a Schiff base.

The imparting or improving of the ion exchange property by the treatment of the porous article of the present invention can be achieved by adjusting the anionic property or cationic property of the polymerized product generated in the porous article. For example, since polymerized ligninsulfonic acid has cation exchange ability, it can be used for plant-based porous articles such as wood, wood-processed materials, wood chips, wood flour, fir, rush, straw, bamboo, fiber, paper, and pulp. It is possible to improve the cation exchange capacity. In order to impart or improve the anion exchange capacity, ο-phenylenediamine, ρ-phenylenediamine, 3,4-diaminobenzophenone, ο-aminophenol, ρ-aminophenol, 1,2-diene An aromatic compound having an amino group such as aminoaminosuraquinone, 1,4-diaminoanthraquinone, a quaternary ammonium salt, polyallylamine, or an aromatic compound having a substituent having a polyethylenimine as a structural part is ligated. And lignin derivatives, or by polymerizing an aromatic amine compound with polyphenoloxidase. The addition or improvement of formaldehyde absorption by the porous article treatment of the present invention can be performed by the sealing ability of the porous article treatment of the present invention. Further, the formaldehyde absorption can be further improved by performing the treatment for imparting or improving the anion exchange ability described above, and reacting the formaldehyde with the amino group in the treating agent. It is also known that polyphenol substances such as catechin or oxides of polyphenol substances deodorize by reacting with malodorous substances such as methyl mercaptan, trimethylamine, ammonia, and tobacco odor. It is useful for deodorizing purposes. The reaction with such a gaseous substance proceeds more efficiently as the surface area of the reaction field is larger. By using it, it is possible to manufacture an article having a high deodorizing effect.

Many conventionally known flame retardants can be used as the chemical for imparting or improving the flame retardancy by treating the porous article of the present invention. For example, Na, K, Mg, C a , Ba, Al, Zn, Cu, Mn, Ni, Si, Sn, Pb, etc. in the cation part, phosphate, hydrogen phosphate, sulfate, sulfate water Salts, carbonates, borates, silicates, nitrates, fluorides, chlorides, bromides, hydroxides, and the like.Specifically, aluminum hydroxide, magnesium hydroxide, zirconium hydroxide, Examples include antimony trioxide, barium metaborate, tin oxide, red phosphorus, and ammonium phosphate. In particular, when ligninsulfonic acid is used as the phenol compound, the alkoxyl group-containing aromatic compound, the halogenated phenol compound, the quinone compound, or the aromatic amine compound of the present invention, the digestion process in the pulp mill is mainly used. As a result of performing with calcium sulfite or magnesium sulfite, the resulting lignin sulfonic acid contains fine powders such as calcium carbonate, calcium hydroxide, magnesium carbonate, or magnesium hydroxide. By treating the porous article of the present invention, these fine powders can be effectively used as a flame retardant. Utilizing the porous article processing of the present invention, for example, manufacturing containers from biodegradable natural materials such as wood chips, wood flour, fir, rush, straw, bamboo, fiber, paper, and pulp Can be. It is desirable to adjust the humidity control, water absorption, water repellency, surface smoothness, biocompatibility, and ion exchange properties of these containers according to the purpose. Further, since the present invention utilizes an enzymatic polymerization method, it is highly safe for the human body and the environment, and the manufactured container can be used in a wide range of fields. It is particularly useful in the field where biodegradability is required in soil, compost, and the like. Even if it is transplanted to the ground as it is, the container will gradually biodegrade, reducing the labor required for transplantation.

The coloring by the treatment of the porous article of the present invention may be performed by polyphenol such as o-phenylenediamine, p-phenylenediamine, catechol, gallic acid, and quercetin. The action of a dye or dye precursor capable of acting on oxidase and polyphenol oxidase on wood to produce a colored substance in wood, or a colored substance and a polyphenol compound such as lignin already contained in wood Is made into a composite polymer in wood, and the wood is strongly dyed and colored. In the above wood dyeing and coloring treatment, it is known that many polyphenol oxidases bleach lignin, which is a coloring substance in wood, and the wood dyeing and coloring treatment of the present invention is carried out enzymatically. Since the bleaching and dyeing / coloring treatments can be performed simultaneously, the process is shortened and the color tone is improved, which is extremely useful. In addition, by using polyphenoloxidase and lignin or lignin derivatives such as ligninsulfonic acid and ligninsulfonate for wood processing, the difference in color tone and chromaticity between the core and sapwood of wood is reduced. Thus, wood having a more uniform and natural texture can be obtained.

Further, the polyphenol oxidase of the present invention is added to concrete together with a phenol compound, an alkoxyl group-containing aromatic compound, a halogenated phenol compound, a quinone compound, or an aromatic amine compound, and these compounds are polymerized in the concrete. It is possible to improve slump loss, improve concrete strength, and control rust on reinforcing steel by lowering oxygen concentration in concrete.

Further, the polyphenol oxidase of the present invention is added to the soil together with a phenol compound, an alkoxyl group-containing aromatic compound, a halogenated funol compound, a quinone compound, or an aromatic amine compound, and these compounds are added in the soil. Maintaining and improving the aggregate structure of the soil, improving water retention, improving the sustained release of antibacterial agents or virus infection inhibitors, improving seed spraying topsoil stability, and improving plant support by molecularization The effect of polymer compound spraying, such as improving the ability to suppress dust generation, improving the ability to prevent soil, sediment, and gravel from collapsing due to wind and rain on ridges and slopes, and improving the fixation of sediment and gravel before laying asphalt Is obtained. Moreover, according to the present invention, it is possible to suppress the viscosity of the spray liquid by setting the polymerization reaction not to be sufficiently advanced at the time of spraying. Handling can be made easier and the penetration of the spray liquid into the underground can be improved.Furthermore, the high molecular weight reaction on the soil surface after spraying makes the spray surface sticky Can be suppressed.

As described above, the treatment agent for the porous article of the present invention may be a composition such as polyphenol oxidase, a phenol compound, an aromatic compound containing an alkoxyl group, a halogenated phenol compound, a quinone compound, an aromatic amine compound, an unsaturated compound, and a drug. Is contained as a constituent, but if necessary, a pH adjuster, a dye, a polymer compound, a specific gravity adjuster, a solid, and the like can be further included.

In the polymerization reaction according to the present invention, an enzyme having a polyphenol oxidizing action is used as an oxidation catalyst, and oxygen in the air can be used as an oxidizing agent, which means that the present invention can be applied to a wide range of applications. Enable. When a large amount of a polymer is produced, it is effective to mechanically stir the reaction solution or to add air or oxygen to the reaction system. In addition, peroxidase and hydrogen peroxide, or an oxidase capable of generating hydrogen peroxide instead of hydrogen peroxide and its substrate are added to the reaction solution, and the reaction of the present invention in which oxygen is used as an oxidizing agent, and hydrogen peroxide is used as an oxidizing agent It is also possible to make the reaction proceed simultaneously.

The treating agent for a porous article of the present invention can be prepared as a single agent by mixing the above composition as a powder or a granulated powder. Granulation is a shaping performed for the purpose of suppressing dust generation, or for the convenience of use depending on the preservability of the treatment agent or the purpose of use, and specifically, granulation of granules and pressing. It can be performed by a granulation operation such as dispensing granulation, fluidized granulation, and centrifugal fluidized granulation. In this case, it is also effective to granulate the enzyme together with the enzyme stabilizer separately from the other treating agent components in order to improve the storage stability of the polyphenol oxidase in the treating agent. Further, the treatment agent for the porous article of the present invention can be prepared by either a high-concentration solution premised on dilution at the time of use or a solution having a concentration that can be used without dilution. In this case, to prevent the treatment agent composition from being oxidized before use, seal the container containing the treatment agent when storing the treatment agent, It is desirable to avoid contact. Also, it is more desirable to suppress the oxidation by using nitrogen gas or an inert gas or degassing during the production of the treating agent. When preparing the treating agent for the porous article of the present invention, a phenolic compound or an aromatic amine compound, and further, a polyphenol oxidase are prepared separately from other treating agent components, and mixed immediately before use. It is also possible to use.

As the oxidizing agent that can be used in the present invention, air, oxygen, ozone, hydrogen peroxide, a hydrogen peroxide precursor, a peracid precursor, or a peracid can be used alone or in combination. Among the oxidizing agents, the hydrogen peroxide precursor dissolves in water to form peroxyl ions. Such substances include monohydrate or tetrahydrate perborates, percarbonates, perborates, sodium perpyrophosphate, perbenzoic acid, urea hydrogen peroxide reactants, melamine hydrogen peroxide reactions And peroxyhydrate, particularly preferred are perborate and percarbonate. Furthermore, a hydrogen peroxide generation system using oxidase and its substrate can be used as a hydrogen peroxide precursor. Examples of such oxidases are glucose oxidase, alcohol oxidase, glycerol oxidase, amine oxidase, amino acid oxidase, D-amino acid oxidase, arylino real oxidase, aldehyde deoxidase, galactose oxidase. And sorbose oxidase, peroxidase, xanthine oxidase, cholesterol rhone oxidase and the like, and particularly preferable are glucose oxidase and alcohol oxidase.

Further, the peracid precursor is an organic compound having a reactive acyl group or a carboxylic acid ester, a carboxylic acid anhydride, or an acetate. Such a substance includes tetraacetylethylenediamine (TAED), Tetraaceti z lemethyleneshimine (TAMDA), tetraacetylglycoluril (TAGU), diacetyldioxohexahydro-triazine (DADHT), xenonanol Sodium sulfonate (sodium nonanoyloxybenzene sulfonate; SNOB S), sodium i sononanoyloxybenzene sulfonate (IS ONOB S), succinic anhydride, benzoic anhydride, phthalic anhydride, There are glucose pentaacetate (glucose pentaacetate; PAG) and xylose tetraacetate, and TAED and SNOBS are particularly preferable.

Further, the peracids include, for example, diperoxydodecanedioic acid (DPDDA), diperoxyisophthalic acid, magnesium monoperoxydiophthalate, and magnesium monohydrate. It is a non-amidoperoxyadipic acid (NAPAA).

[Other usage methods]

Flavonoid, xanthone, melanin and other plant pigments and lignin are known as natural products having polyphenol in the structural part, and polyphenol oxidase has an oxidizing effect on these natural products. Polyphenol oxidase can also be used as a reaction substrate for halogen-substituted phenols (AOX), such as phenol or trichlorophenol, for which toxicity is a problem. Therefore, for example, the polyphenol oxidase of the present invention is also useful for treating wastewater containing these natural products and non-natural products. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, representative examples of the present invention will be described, and the present invention will be described more specifically. However, these are merely examples, and the present invention is not limited to these. Further, in the following examples, “%” means “% by weight” unless otherwise specified. Example 1: Culture and concentration

Glucose (1%) and 4 2 liter flasks _{NH 4 C 1 (0.2%)} , N a 2 HP0 4 - 1 2H 2 0 (1.34%), KH 2 P0 4 (0.3%), Na C l (0.1%), peptone (0.2%), Mg S 0 _A 500 ml medium containing 4.7 H ₂ 0 (0.05%) and CuSO 4 (0.02 mM) was added to 2 N--NaOH to adjust the pH to 7.5, and the mixture was added to each flask. It was prepared and inoculated with Stilbella sp. (Stilbella sp.) SD3101 (Accession No. FERM BP-6219), followed by shaking culture at 27 ° C for 100 hours. After the culture, the culture broth was removed by centrifugation at 4 ° C. Next, the culture broth that had been sterilized was concentrated as a fraction with a molecular weight of 10,000 or more by a minitan ultrafiltration system (Millipore) using a minitan filter packet (CAT.N0: PTGC0MP04, manufactured by Millipore). did.

Example 2: Crude purification

_{_{N a 2 ΗΡ0 4 · 1 2Η}} 2 0 (1.34%), ΚΗ 2 Ρ0 4 (0.3%), N a C l equilibrated with (0.1%) was Jeffrey chill aminoethyl cellulose (DEAE) -Cellulofine A- 800m (raw Chemical Industry Co., Ltd.) The concentrated culture broth described in Example 1 was applied to the top of the column (06 Omm, 330 cc) in the space above the column, and a buffer of the same composition as that used for equilibration. The column was washed with 400 mH. Then, N a ₂ HP_〇 _{_{4 · 1 2 H 2 O (}} 1.34%), KH 2 P0 4 (0.3%), using One not a 5 Om l once the Na C l (0.3%), total After eight elutions, polyphenoloxidase activity was obtained in the second to fifth fractions. These active fractions were concentrated by ultrafiltration system as described in Example 1, further, after dialysis against NH ₄ HC_〇 ₃ (200 p pm), subjected to freeze-drying, freezing the crude product Obtained as a dried product. The polyphenol oxidase activity of the lyophilized product was 5 U / mg. Example 3: Culture and concentration, crude purification

(Sagenomella sp.) SD3102 (Accession No. FERM BP-6220) was cultured and concentrated in the same manner as in Example 1 to obtain an aqueous solution having polyphenoxylidase activity. Further, the same crude purification as in Example 2 was performed to obtain a lyophilized product. The lyophilizate had a polyphenoloxidase activity of 3.5 U / mg. Example 4: Culture and concentration, crude purification

(Stachylidium sp.) SD3103 (Accession No. FERM BP-6221) was cultured and concentrated in the same manner as in Example 1 to obtain an aqueous solution having a polyphenol oxidase activity.

Further, the same crude purification as in Example 2 was performed to obtain a lyophilized product. The lyophilizate had a polyphenoloxidase activity of 2.5 U / mg. Example 5: Measurement of isoelectric point

Using the polyphenoloxidase activity samples obtained in Examples 2 to 4, the activity staining and isoelectric point were measured by isoelectric focusing using an oral Tofoa 'system (BIO-RAD¾i¾). pH 2.5 to 5) (Sigma) The activity of the gel plate after the electrophoresis was performed using 50 ppm of syringaldorezine and 10 OmM. It was immersed in an aqueous solution containing a potassium phosphate buffer solution (pH 8.2), and observed by observing the red-purple band resulting from the reaction with polyphenol oxidase.

As a result, Stilbella sp. (Stilbella sp.) SD3101 (Accession number FERM BP-6219), Sagenomella sp. (Sagenomella sp.) SD3102 (Accession number FERM BP-6220) ), Stachylidium 's p. (Stachylidium sp.) SD3103 (Accession No.FERM BP-6221) has isoelectric points of 6.6 ± 0.5, 5.9 ± 0.5 and 7.0 ± 0.5, respectively. there were. Example 6: Substrate specificity

Substrate specificity of the oxidation reaction was examined using the partially purified polyphenol oxidase described in Examples 2 to 4. At room temperature (25 ° C), 0.05 mM substrate and 0.1 M P

Of oxygen consumption rate between solutions with and without enzyme in potassium phosphate buffer (pH 8.0) using a manometer (YSI Model 5300 Biological Oxygen Monitor Yellow Springs Instrument o., Inc.) It was done by doing something. In this measurement, the difference between the oxygen consumption rates when ascorbic acid was used as the substrate (Δ oxygen consumption rate) was the largest. As a control, the results obtained when the same measurement was performed using commercially available ascorbate oxidase (derived from Cucumis sp., Manufactured by Funakoshi Co., Ltd.) are also shown.

Example 7: molecular weight

The molecular weight was measured using gel filtration chromatography (GFC). _{_{_{Na 2 HP0 4 'l 2H 2}}} 0 (1.34%), KH 2 P 0 4 (0.3%), GFC column equilibrated at a flow rate of 1.0 m 1 / min by Na C l (1%) ( Shodex PROTEIN KW- 802.5 , Two columns) and an HP detector using a UV detector (280 nm), the crude polyphenol oxidase described in Examples 2 to 4 was analyzed, fractionated, and assayed for polyphenol oxidase. The activity peaks were eluted at the molecular weights of 76,000 ± 5000, 33,000 ± 5000, and 55,000 ± 5000, respectively. As a molecular weight marker protein, MW of Oriental Industry Co., Ltd. P

— A Marker (HP LC) was used. Example 8: Cultivation and crude purification in a 5-liter flask

Glucose _(1/0) and _{NH 4 C 1 (0.2%)} , N a 2 ΗΡ0 4 · 1 2 Η 2 0 (1.34%), Κ Η 2 Ρ Ο 4 (0.3%), Ν a C 1 (0.1 %), peptidase tons _{(0.2%), Mg S0 4} - 7H 2 0 (0.05%), Cu S_〇 ₄ (0.02 mM) to the medium of Tona Ru 1 liters 2 N-N 7.5 is added to bring the pH a OH (Stilbella sp.) SD3101 (Accession number FERM BP-6219) was inoculated into two 5-liter flasks containing the following, and shaken at 28 ° C for 5 days. Culture was performed. After culturing, 1.8 liters of the culture broth was removed by centrifugation at 4 ° C.

Next, a part of this culture broth was subjected to fractionation with a molecular weight of 10,000 or more by a minitan ultrafiltration system (Millipore) using Minitan 'filter packet (CAT. NO .: PTGC0MP04, manufactured by Millipore). And concentrated. This was further subjected to DEAE-Cellulofine A-80 Om column chromatography, and the eluted active fraction was again concentrated by the above-mentioned minitan ultrafiltration system to a fraction having a molecular weight of 10,000 or more. After a further dialysis against NH ₄ HC0 ₃ The re-concentrate (200 p pm), subjected to freeze-drying to obtain a crude product as a freeze-dried product. The lyophilized product had a polyphenoloxidase activity of 8 UZmg.

In addition, by the same culture and crude purification operations, Sagenomella sp. (Sagenomella sp.) SD 3102 (Accession number FERM BP-6220) and Stachyridium sp. (Stachvlidium sp.) SD 3103 (Accession number FERM BP A freeze-dried product of porifenol oxidase produced by -6221) was obtained. The activities were 5 U / mg and 4.5 U / mg, respectively. Example 9: Mixing with conventional polyphenoloxidase

A commercially available polyphenoloxidase (Rigidoporus zonal is derived, obtained from TaKaRa Co., Ltd.) and SD3101-SD3103 (lyophilized product) described in Example 8 and the activity was measured at various pH values. However, as shown in FIG. 11, the oxidation reaction proceeded rapidly in a wide range of pH from pH 4 to 10. Example 10: Polymerization reaction

Lignin sulfonic acid sodium salt (obtained from Aldrich Chemical Company, Inc.), 30% (W / V), as polyphenoloxidase, was used as Stilbella's sp. sp.) Prepare 1 ml of a reaction solution containing a freeze-dried product obtained from SD3101 (Accession number FE thigh BP-6219) at an active concentration of 300 UZ liter, and react in a glass test tube. The reaction was carried out by shaking at 100 rpm at a temperature of 30 ° C. The pH of the reaction solution was adjusted to 8.2 with a small amount of sulfuric acid. Immediately after the start of the reaction, the color tone of the reaction solution became deep, and after 8 hours, remarkable progress of polymerization was observed, and after 20 hours, most of the reaction solution was solidified.

(Sagenomella sp.) SD3 102 (Accession number FERM BP-6220) or stachyridium sp.

(Stachvlidium sp.) When the reaction was carried out in the same manner as in the above example using the freeze-dried product obtained from SD3103 (Accession number FERM BP-6221), the polymerization and solidification proceeded at the same rate. .

In addition, lignin (alkali) (obtained from nacalai tesque) was used in place of ligninsulfonic acid at a concentration of 30% (W / V), and the activity concentration of polyphenol oxidase at 300 U / liter and pH 8.5 was used. When the reaction was carried out, as a result of the progress of the polymerization reaction, the entire reaction solution was a highly viscous liquid 24 hours after the start of the reaction.

The sample for molecular weight analysis was sampled from the reaction solution, subjected to a heat treatment at about 100 ° C. for 15 minutes in a water bath to stop the reaction, and analyzed by HP LC. Analysis by HP LC was performed using 5 OmM phosphoric acid as eluent. Buffer (pH 7.0) or 0. ImM sodium sulfate aqueous solution, a differential refractive index detector (Shodex RI, manufactured by Showa Denko KK) was used as the detector, and Shodex PROTEIN KW-802.5 (Showa This was performed by GFC using a combination of Shodex PROTEIN KW802.5 and Shodex OHpak SB-804HQ (manufactured by Showa Denko KK).

Reaction containing 30% (W / V) ligninsulfonic acid sodium salt and commercially available polyphenol oxidase (derived from Rigidoporus zonalis, obtained from TaKaRa Co., Ltd.) as polyphenol oxidase at an active concentration of 300 UZL 1 ml of the liquid was prepared, and the reaction was carried out in a glass test tube by shaking at a reaction temperature of 30 ° C. and 100 rpm. The commercially available polyphenol oxidase used here was shown to be an acidic enzyme having an optimal reaction pH of pH 6 to 7 in the activity measurement using syringaldazine, so that it was used in the polymerization reaction. The pH of the reaction solution was adjusted to 6.5 using a small amount of sulfuric acid. Immediately after the start of the reaction, the color tone of the reaction solution became dark, but it took about 80 hours for the most of the reaction solution to solidify. When this enzyme was used in a similar polymerization reaction with the pH of the reaction solution at 8.5, no solidification was observed even 80 hours after the start of the reaction. Example 11 1: Soil modification

Lignin sulfonic acid sodium salt at 30% (W / V), polyphenol oxidase, as described in Example 8, stillbella.Sp freeze (Stilbella sp.) SD 3101 (accession number FERM BP-6219). At a reaction temperature of 30 ° C and pH 8.5 to obtain a water-soluble polymerized lignin sulfonic acid, which was diluted twice with water. Was prepared. The viscosity of the solution before dilution was measured with an R-type viscometer (VISC0NIC EHD R (R0TER No.7), manufactured by Tokyo Keiki Co., Ltd.). At a shear rate of 1, 16 Pa · s, shear It was 14.5 Pa · s at a speed of 2 and 11.25 Pa · s at a shear rate of 20. The operation of stopping the polymerization reaction by heat or the like was not performed. This polymerized lignin sulfonic acid aqueous solution 3.Om1 or water 3.Om1 was sprayed onto the soil surface in a 5Om1 glass beaker containing field soil (40 g) and incubated at 28 ° C. The weight loss due to drying was measured. After 60 hours, the weight of the polymerized lignin sulfonic acid solution was reduced by about 8 g, and the weight of the water sprayed water was reduced by about 11 g. Admitted. Sprayed with a polymerized lignin sulfonic acid solution also shows an improvement in the hardness of the soil surface, which is useful for applications such as maintaining the aggregate structure, modifying the soil, and stabilizing the topsoil of seed spraying. It was shown that.

(Sagenomella sp.) SD 3102 (Accession No. FERM BP-6220) or stachyridium sp.

(Stachylidium sp.) When the reaction was carried out in the same manner as in the above example using the freeze-dried product obtained from SD3103 (Accession number FERM BP-6221), the same improvement in water retention and hardness of the soil surface The improvement was recognized. Example 12 2: Wood treatment

A 1% aqueous solution of p-phenylenediamine 'dihydrochloride (manufactured by Kanto Chemical Co., Ltd.) was adjusted to pH 8.5 with a small amount of NaOH, and was used as a polyphenol oxide as described in Example 8. Stilbella sp. (Stilbella sp.) A freeze-dried product obtained from SD3101 (Accession No. FERM BP-6219) was added at an activity concentration of 2 OUZ liters, and immediately the cedar wood chips (3 The pressure was reduced at 650 to 700 mmHg for 30 minutes with the cmX 3 cmX lcm and the kiguchi surface 3 cmX 3 cm) submerged, and the sample was left at normal pressure for 15 minutes while immersed. Thereafter, a piece of wood was taken out, the solution on the surface was wiped off, and left at 24 ° C. for 2 days to allow drying and enzymatic reaction to proceed.

Furthermore, 20 Om 1 of water was added to the piece of wood, and the rotor was rotated with a magnetic stirrer at 25 ° C for 8 hours while the piece of wood was submerged under water. Was wiped off and left at 24 ° C. for 4 days to dry. In the wood piece thus obtained, the coloring reaction rapidly progressed on the surface and inside of the wood piece, and as a result of depositing a black-brown colored material, the inside of the wood piece was also strongly colored. Example 13: Wood treatment

Wood containing polyphenoloxidase (freeze-dried product) (300 UZ liter) described in Example 8, ligninsulfonic acid sodium salt (5%, W / V), copper sulfate (0.04M), and ethylenediamine (0.08M) A reaction solution for the treatment was prepared, and the cedar wood pieces (3 cm × 3 cm × 2 cm, 3 cm × 3 cm on the tip) were subjected to vacuum impregnation. The pH was adjusted with sodium hydroxide or sulfuric acid.

The vacuum impregnation operation was performed by a simple method in which cedar wood pieces were immersed in the treatment solution, then reduced at 65-70 OmmHg for 1 hour, and then kept at normal pressure for 30 minutes while immersed. It was confirmed by measurement of the weight of the wood chips before and after the impregnation operation that a sufficient amount of the treatment liquid (10 to 14 g) had been injected by this vacuum impregnation operation.

Furthermore, the impregnated wood pieces were placed in a constant temperature room at 28 ° C for 5 days, and after drying and high-molecularization reaction, 20 Oml of water was added to each wood piece and the wood piece was submerged under the water surface The leaching operation was performed by rotating the rotor using a magnetic stirrer and stirring at 25 ± 3 ° C for 8 hours. Then, by measuring the absorbance at 280 nm of the water (leaching solution) after the leaching operation, the leaching amount of ligninsulfonic acid can be determined. Pyridylazo) -2-naphthol; abbreviated as PAN. The leaching amount of the drug was calculated by performing complex formation and absorption analysis using Aldrich (Chemical) Company). The results showed that wood pressure treatment using polyphenol oxidase and ligninsulfonic acid enabled effective drug injection and immobilization, and that the effect was particularly large on the side of pH 8 or higher. . For comparison, detailed results are shown in Table 2, together with the results in the absence of polyphenol oxidase. Table 2

LSA; Likuninsurenoleic acid

: EDA; Echirendiamine Example 14: Wood treatment

(Stilbella sp.) SD3101 (Accession No. FERM BP-6219) -derived polyphenol oxidase (lyophilized product) described in Example 8

A reaction solution for wood treatment containing 300 U / liter, and 5% (W / V) of sodium ligninsulfonate and various chemicals was prepared, and a cedar wood chip was prepared in the same manner as in Example 13. Vacuum impregnation treatment, drying and polymerization reaction, and leaching treatment were performed. For drugs with low water solubility, lignin sulfonic acid aqueous solution is

After heating to 90 ° C, the drug is added, and the drug is suspended, dispersed or dissolved by a vortex mixer, then cooled to 25 ° C, and the polyphenol is added. A treatment liquid was prepared by adding a freeze-dried product of luoxidase.

Then, by measuring the absorbance at 28 O nm of the water (leaching solution) after the leaching operation, the leaching amount of ligninsulfonic acid, and PAN or quinalizarin (manufactured by Wako Pure Chemical Industries, Ltd.) The amount of drug leached was calculated by complexation and absorption spectroscopy using HPLC, or separation, identification, quantification, or atomic absorption analysis using HPLC or gas chromatography. If the drug used has an absorbance at 280 nm, calculate the absorbance at 280 nm of the drug whose concentration was measured by the above method, and calculate the absorbance at 280 nm of the leached solution. The concentration of lignin sulfonic acid was calculated by dividing the influence component. The amounts of lignin sulfonic acid and drug injected during the impregnation treatment were set at 100%, and the leached amounts were compared. The results showed that wood pressure treatment using polyphenol oxidase and ligninsulfonic acid enabled effective drug injection and fixation, and that the effect was particularly great on the alkaline side at pH 8 or higher. For comparison, Table 3 shows the detailed results, together with the results in the absence of polyphenol oxidase. Incidentally, (+) - Power catechin & Eta ₂ 〇, tannic acid, Ariru isothiocyanate Xia sulphonate from Company, have Aldori pitch-Kemikano, copper carbonate (copper (II) carbonate monohydrate), copper sulfate, Zinc, nickel chloride, boric acid, and silver sulfate were obtained from Wako Pure Chemical Industries, Ltd., and hinokitiol and 1,3,5-tritalirololbenzene were obtained from Tokyo Chemical Industry Co., Ltd., respectively.

P

Table 3

EDA; ethylenediamine

Η ； Hinoki Chionore

Τ; Tween80 used at a concentration of 500 ppm Example 15: Wood treatment

As the polyphenol oxidase system, the Sagenomera sp. Described in Example 8 was used.

(Sagenomella sp.) SD 31 O 2 (Accession No. FERM BP-6220) polyphenol oxidase (lyophilized product) obtained from 30 O UZ liter, sales peroxy Using 5,000 UZ liter of dase, 5000 U / liter of alcohol oxidase, and 1% of methanol, the cedar wood chips were subjected to vacuum impregnation, drying, polymerisation, and leaching in the same manner as in Example 13. The amounts of ligninsulfonic acid and drug injected during the impregnation treatment were set at 100%, and the leached amounts were compared. The results showed that similarly effective drug injection and fixation was possible. Table 4 shows the detailed results. Peroxidase used was a horseradish-derived product (Type II, manufactured by Sigma), and alcoholoxidase used was a product derived from Candida boidini (manufactured by Boehringer Mannheim Biochemica).

Table 4

EDA; ethylenediamine

ヒ; Hinokitiol Example 16: Wood treatment

Stilbella sp. (Stilbella sp.) SD3 101 (Accession No. FERM BP-6219) described in Example 8 (300 liters of polyphenol oxidase (lyophilized product)) and 5% of ligninsulfonic acid sodium salt (W / V), and a wood treatment reaction solution containing 2000 ppm of tung oil was prepared.Similar to Example 13, the cedar wood chips were subjected to vacuum impregnation treatment, drying, polymerization reaction, and leaching treatment. The amounts of lignin sulfonic acid and drug injected were set at 100%, and the leached amounts were compared. As a result, a further improvement in drug fixability was observed. Details JP9

Table 5 shows the results.

Table 5

EDA; --Η; Hinokitiol

Τ; Tween80 used at a concentration of 500 ppm Example 17: Wood treatment

10 g of rust rhizome was crushed by a mixer, 10 ml of water was added, and this was filtered through a cloth to obtain a rust extract. (Stilbella sp.) SD3101 (Accession No. FERM BP-6219) -derived polyphenoloxidase (freeze-dried product) described in Example 8 was used at 30 OU / liter and lignin sulfone. A reaction solution for wood treatment containing 5% (W / V) of sodium acid salt and 5% of the above rust extract was prepared, and the cedar wood chips were subjected to reduced pressure impregnation treatment, drying and polymer as in Example 13. The leaching amount was calculated based on the amount of lignin sulfonic acid injected during the impregnation treatment as 100%, and as a result of the progress of the polymerization reaction inside the wood, the leaching amount of lignin sulfonic acid was determined. Was only 5.8%, indicating that a wood treatment containing rust extract was obtained. Example 18: Wood treatment

(Stilbella sp.) SD3101 (Accession No. FERM BP-6219) -derived polyfunoroxidase (lyophilized product) described in Example 8 Prepare a reaction solution for wood treatment containing 300 U / liter, 5% (W / V) ligninsulfonic acid sodium salt and various other chemicals, and apply cedar wood chips (3 cmX 3 cmX) as in Examples 3 and 4. Decompression (pre-evacuation) and pressure impregnation were performed on 2 cm, 3 cm x 3 cm on the wood front.

For depressurization and pressurization, place a cedar wood piece in a pressure reactor (3 liter capacity), put a weight on the wood piece to sink the wood piece, and use a vacuum pump to exhaust 600 720 mmHg for 30 minutes. After that, the wood is immersed in the processing liquid by flowing the processing liquid using the negative pressure inside the reactor, and a pump (T-50K TEST-PUMP, Kyo Co., Ltd.) The processing solution was flowed in using a pressure of 10 atm for 1 hour.

The wood chips after the impregnation treatment were further dried, polymerized, and leached in the same manner as in Example 13. Compared. The results showed that equally effective drug injection and fixation was possible. Table 6 shows the detailed results.

Table 6

EDA; ethylenediamine

H: Hinokitiol Example 19: Wood treatment

Stilbella sp. (Stilbella sp.) SD31O1 (received A reaction solution for wood treatment containing polyphenoloxidase (freeze-dried product) derived from Deposit No. FERM BP-6219) containing 3 OUZ liter and ligninsulfonic acid sodium salt at 2% (W / V) was prepared. In the same manner as in 18, the cedar wood chips were subjected to pressure reduction and pressure impregnation, drying, and polymerization reaction. The pH of the reaction solution was adjusted to 8.5. Next, an aqueous solution containing 0.4% (W / V) polyethyleneimine (Aldrich 'Chemical Co., Ltd., average molecular weight 700) and 0.02M copper sulfate was prepared. The same impregnation treatment under reduced pressure and pressure was performed to perform the second impregnation operation. After air-drying the treated wood obtained in this way for 6 days, the same leaching treatment as in Example 13 was performed, and the amount of leaching was calculated with the amount of copper ions injected at the time of impregnation as 100%. The leaching amount was only 1.5%. It was shown that the polymerization and fixation of ligninsulfonic acid inside the wood and the formation of a complex of ligninsulfuric acid, copper ions and polyethyleneimine caused the copper ion to be firmly fixed in the wood.

As a first step, an aqueous solution containing 5% (W / V) polyethyleneimine and 0.25 M copper sulfate was injected into cedar wood chips in the same manner as in Example 13 and air-dried for 6 days. Example 3 Stilbella sp. (Stilbella sp.) SD3101 (accession number FERM BP-6219) derived polyfunoxoxidase (lyophilized product) was collected at 300 U / liter and sodium ligninsulfonate. A reaction solution containing 10% (W / V) of a lithium salt was applied to the piece of wood, and placed in a constant temperature and humidity incubator at 28 ° C and a relative humidity of 80% for 3 days to allow the enzyme polymerization reaction to proceed. The resulting wood chips prevented the copper complex from migrating to the wood surface and had a more natural brown surface color. Example 20: Wood treatment

Stilbella sp. Described in Example 8 (Stilbella sp.) SD31 01 (Accession No. FERM BP-6219) derived from polyfunoxoxidase (lyophilized product) in 300 UZ liter and ligninsulfonic acid sodium salt in 5% (W / V), and a tree containing 0.5% of p-phenylenediamine dihydrochloride (Kanto Chemical Co., Ltd.) A reaction solution for wood treatment was prepared, and depressurization was carried out on a piece of cedar wood (3 cm x 3 cm x 10 cm, 3 cm x 3 cm at the tip end) partially containing a core material. And a pressure impregnation treatment, followed by the same drying and polymerizing reaction and leaching treatment as in Example 13. As a result, the sapwood portion of the wood was more strongly impregnated, developed, and fixed. The difference in color tone and chromaticity between the core material and the sapwood became small, and a brown-colored wood having a more uniform appearance could be obtained. Example 21: Antibacterial test of processed wood

Using a 500 ml glass beaker as an incubator, 100 ml of agar medium containing glucose (4%), 1.5% malt extract, 0.3% peptone and 2% agar ( pH 6.5) was inoculated with the rot fungus Tyromyces palustris FEPRI 0507 or the viper mushroom (Coriolus versicolor) FEPRI 1030 (both obtained from the Ministry of Agriculture, Forestry and Fisheries, Forestry Research Institute) at 26 ° C and 1 ° C. A week of culture was performed. On the thus obtained culture, the leached wood chips obtained in Example 18 were directly sterilized with agaric mushrooms and sterilized with a heat-resistant plastic having a thickness of about 1 mm with mushroom mushrooms. An antibacterial operation was performed by placing a net and placing the fiber vertically on it, and placing it at 26 ° C for 12 weeks. Before the antibacterial operation, the leached wood chips obtained in Example 9 were placed in a dryer at 60 ° C for 48 hours, and then placed in a desiccator for 30 minutes to be sufficiently dried. After that, the weight before the antibacterial operation was measured. After the antibacterial operation, remove the wood pieces from the incubator, remove the mycelium on the surface sufficiently, air-dry for about 24 hours, and then dry them sufficiently using a dryer and desiccator as described above, and measure the weight. Then, the weight loss rate was calculated in comparison with the weight before the antibacterial operation. As a result, it was shown that the wood treatment method of the present invention can impart antibacterial properties. Table 7 shows the detailed results. Table 7

Wood treatment conditions Weight loss rate (%)

EDA; Ethylenediamine Example 22 Test for termite control of wood products

The dry weight of the leached wood chips obtained in Example 18 was measured in the same manner as in Example 21, and these were placed on soil about 40 cm around the termite nest. After standing for months, the effect of termites was observed. The soil on the surface was sufficiently removed, and the dry weight was measured in the same manner as in Example 21. The weight loss was calculated by comparing the dry weight with the weight before installation. As a result, it was shown that the wood treatment method of the present invention can impart termite resistance. Table 8 shows the detailed results. '

Table 8

EDA; Ethylenediamine Example 23: Porous article treating agent

Stilbella sp. (Stilbella sp.) SD3101 (accession number FERM BP-6219) -derived polyphenoloxidase (lyophilized product 12 mg) described in Example 8 and ligninsulfonic acid sodium salt powder ( To 10 g), various drugs were further added and mixed well using a mortar. Furthermore, when a 5% aqueous solution is prepared using this powder, a small amount of sodium carbonate powder is added as necessary so that the pH is 8.0 to 9.0, and the mixture is thoroughly mixed to obtain a powdery porous material. A quality article treating agent was obtained. Next, the powdered treatment agent is left in a glass container at room temperature for 2 weeks, and the powder (5 g) is dissolved in ion-exchanged water (lOOml) to form a porous article. A solution for processing was prepared. Using this solution, the cedar wood chips were subjected to depressurization and pressure impregnation, drying and polymerizing reaction, and leaching as in Example 13, and the amounts of ligninsulfonic acid and chemicals injected during the impregnation were determined. The leaching amount was compared at 100%. As a result, it was shown that the porous article can be effectively treated by the powdery treating agent. Table 9 shows the detailed results.

The powdery treating agent (6 g) obtained in the same manner as above was dissolved in ion-exchanged water (12 ml) to obtain a solution-like porous article treating agent. Next, this solution processing The agent was transferred to a 20 ml screw cap test tube, left at room temperature for 2 weeks in a sealed state, and further diluted 10 times with ion-exchanged water to prepare a solution for treating porous articles. Using this solution, the cedar wood chips were subjected to depressurization and pressure impregnation, drying, polymerization reaction, and leaching as in Example 13, and the amounts of ligninsulfonic acid and chemicals injected during the impregnation were determined. The amount of leaching was compared with 100%. As a result, it was shown that the porous article can be effectively treated even with the solution treating agent. Table 10 shows the detailed results.

Table 9

EDA; ethylenediamine

H: Hinokitiol

Table 10

EDA; ethylenediamine

H: Hinokitiol

Industrial applicability

As described above in detail, according to the present invention, an efficient polymerization reaction of a phenol compound, an alkoxy group-containing aromatic compound, a halogenated phenol compound, a quinone compound, or an aromatic amine compound is achieved. , Its thickeners containing polymeric compounds, stabilizers, flocculants, emulsifiers, dispersants, water retention agents, antioxidants, adhesives, concrete admixtures, dyes, paints, oil recovery agents, soil modifiers , Seed spraying topsoil stabilizer, sealant, deodorant, deodorant, pesticide spreading agent, bactericide, antibacterial agent, virus infection inhibitor, biofouling inhibitor, biological repellent, insecticide, cataplasm, An ink base and a wood treating agent are provided efficiently.

Further, by utilizing the present invention, a thickener, a stabilizer, a step of polymerizing a phenol compound, an alkoxyl group-containing aromatic compound, a halogenated phenol compound, a quinone compound, or an aromatic amine compound, Flocculant, emulsifier, dispersant, water retention agent, antioxidant, adhesive, concrete admixture, dye, Paints, oil recovery agents, soil modifiers, seed spraying topsoil stabilizers, sealants, deodorants, deodorants, pesticide spreading agents, bactericides, antibacterial agents, virus infection inhibitors, biofouling inhibitors, organisms An efficient method for producing repellents, insecticides, cataplasms, ink bases and wood treatments is provided.

Further, by using the polyphenol oxidase of the present invention, an effective method for wood treatment, concrete treatment, soil treatment and the like is provided.

Claims

The scope of the claims

1. The following characteristics:

(1) Action: oxidizes polyphenol;

(2) Optimum reaction pH: ∑) Has an optimum reaction 11 around ^ 18.2 to 8.5;

(4) molecular weight: the molecular weight measured by gel filtration chromatography (GFC) analysis is in the range of about 33,000 to 76,000 內;

A method for polymerizing a phenolic compound, an alkoxyl group-containing aromatic compound, a halogenated phenolic compound, a quinone compound, or an aromatic amine compound, characterized by reacting a polyphenoloxidase having the following formula:

2. The method according to claim 1, wherein polyphenol oxidase is allowed to act in an alkaline region having a pH of 8 or more.

3. The method according to claim 1, wherein polyphenoloxidase is used together with a substance having a peroxidase activity.

4. The method according to any one of claims 1 to 3, wherein polyphenol oxidase is used together with the oxidase and its substrate.

5. The method according to any one of claims 1 to 4, wherein air, oxygen, ozone, hydrogen peroxide, hydrogen peroxide precursor, peracid precursor or peracid is used alone or in combination as an oxidizing agent. Polymerization method.

6. The following characteristics: