JP2002356405A - Method for wood treatment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method in which a phenolic compound or an aromatic amine compound is efficiently polymerized by an enzyme catalyst in an alkali range and the polymerization reaction is applied to wood treatment. SOLUTION: This method for wood treatment is characterized in that an enzyme having a polyphenol oxidation action and a phenolic compound or an aromatic amine compound are impregnated into wood in an alkali pH range and the phenolic compound or the aromatic amine compound is polymerized in wood.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for enzymatically polymerizing a phenolic compound or an aromatic amine compound and a method for using the polymer compound obtained by the method.
More specifically, the present invention provides a method for producing a phenolic compound or aromatic amine compound having an increased molecular weight by causing an enzyme having a polyphenol oxidizing action to act on a phenolic compound or aromatic amine compound in an alkaline pH range. And a thickener, a stabilizer, a flocculant, an emulsifier, obtained by utilizing a rapid increase in the molecular weight of a phenolic compound or an aromatic amine compound utilizing the catalytic action of an enzyme in the alkaline region.
Dispersant, water retention agent, antioxidant, adhesive, concrete admixture, dye, deodorant, paint, oil recovery agent, soil modifier, seed spraying topsoil stabilizer, pesticide spreading agent, feed binder, sterilization Agent, antibacterial agent, virus infection inhibitor, biofouling inhibitor, biological repellent, insecticide, cataplasm, ink base and wood treatment agent, production method of these various agents, wastewater treatment method, deoxygenation method, wood A treatment method, a concrete treatment method, and a soil treatment method are provided.

[0002]

2. Description of the Related Art Conventionally, phenolic compounds and the like have been
For example, using a laccase or polyphenol oxidase produced by Basidiomycetes and Deuteromycetes are known to be polymerized (Journal of Biotechnology, 13, 22
9-241, 1990). However, since the optimum reaction pH of laccase and polyphenol oxidase produced by fungi is in the acidic range, in order to catalyze and accelerate the polymerization reaction using these enzymes, the reaction must be carried out at an acidic to neutral pH. And the speed of the polymerization reaction was not sufficiently high. In addition, many of the natural organic compounds that can react with these enzymes are polyphenol compounds. Although the solubility of these polyphenol compounds decreases in the acidic to neutral pH range, the optimal reaction pH of the enzyme is in the acidic region. Because
It is necessary to carry out the reaction in an acidic to neutral pH range, and there is a disadvantage that a high-concentration polyphenol compound cannot be efficiently polymerized. In addition, despite the auto-oxidation of many polyphenol compounds accelerated in the alkaline pH range, the conventional method of enzymatic oxidative polymerization in the acidic to neutral pH range effectively utilizes auto-oxidation. There was a disadvantage that it could not be done.

Further, it has been known that phenolic compounds and the like can be polymerized by bilirubin oxidase and can be used for polymerizing lignin and dyeing cotton (WO95-01426, JP-A-6-316874).
However, in the prior art using bilirubin oxidase, enzyme-catalyzed polymerization of phenolic compounds and the like is performed in an acidic to neutral pH range, and the rate of the polymerization reaction is not sufficiently high. There is no description suggesting that the polymerization reaction of the phenolic compound is accelerated in an alkaline pH range by the method.

[0004]

Accordingly, an object of the present invention is to provide a method using an enzyme catalyst for polymerizing a phenolic compound or an aromatic amine compound in an alkaline pH range.

Another object of the present invention is to provide a thickener and a stabilizer comprising a step of efficiently polymerizing a phenolic compound or an aromatic amine compound in an alkaline pH range using an enzyme having a polyphenol oxidizing action. , Flocculants, emulsifiers, dispersants, water retention agents, antioxidants, adhesives, concrete admixtures, dyes, paints, oil recovery agents, soil modifiers,
Seed spray topsoil stabilizer, deodorant, deodorant, pesticide spreading agent, feed binder, bactericide, antibacterial agent, virus infection inhibitor, biofouling inhibitor, biorepellent, insecticide, cataplasm,
An object of the present invention is to provide a method for producing an ink base and a wood treatment agent.

Another object of the present invention is to provide a thickener, a stabilizer, a coagulant, an emulsifier, an emulsifier, a dispersant, and a water-holding agent containing a polymer compound obtained by efficiently polymerizing the phenolic compound or aromatic amine compound. Agents, antioxidants, adhesives, concrete admixtures, dyes, paints, oil recovery agents, soil modifiers,
Seed spray topsoil stabilizer, deodorant, deodorant, pesticide spreading agent, feed binder, bactericide, antibacterial agent, virus infection inhibitor, biofouling inhibitor, biorepellent, insecticide, cataplasm,
An object of the present invention is to provide an ink base and a wood treatment agent.

Another object of the present invention is to provide a method for removing a compound from wastewater containing a phenolic compound or an aromatic amine compound by utilizing the polymerization reaction according to the present invention. is there. Another object of the present invention is to provide a deoxygenation method for removing dissolved oxygen by utilizing the polymerization reaction according to the present invention.

Another object of the present invention is to provide a method for treating wood utilizing the polymerization reaction according to the present invention. It is another object of the present invention to provide a method for treating concrete using the polymerization reaction according to the present invention. It is a further object of the present invention to provide a soil modification method using the polymerizing reaction according to the present invention.

[0009]

Means for Solving the Problems The present inventors have made intensive studies to develop a method for efficiently polymerizing a phenolic compound or an aromatic amine compound. Surprisingly, it has been found that the use of a suitable enzyme having a polyphenol oxidizing action in an alkaline region, particularly an alkaline pH region of pH 8 or higher, enables efficient polymerization of a phenolic compound or an aromatic amine compound. Thus, the present invention has been completed.

[0010] The present invention relates to a method of treating wood having the following constitution, among the above-mentioned methods of utilizing the method of polymerizing. 1. A wood treatment method comprising impregnating wood with an enzyme having a polyphenol oxidizing action together with a phenolic compound or an aromatic amine compound in an alkaline pH range, and polymerizing the phenolic compound or the aromatic amine compound in the wood. . 2. 2. The wood treatment method according to the above 1, wherein the wood is polymerized in an alkaline region having a pH of 8 or more. 3. 3. The wood treatment method according to 1 or 2, wherein catechol oxidase, laccase, polyphenol oxidase, ascorbate oxidase, or bilirubin oxidase is used alone or in combination as an enzyme having a polyphenol oxidizing action. 4. 4. The wood treatment method according to any one of 1 to 3, wherein the enzyme having a polyphenol oxidizing action is an enzyme obtained by culturing a bacterium belonging to the genus Bacillus. 5. 5. The method for treating wood according to 4 above, wherein the bacterium belonging to the genus Bacillus is Bacillus licheniformis or Bacillus natto. 6. The wood treatment method according to the above item 5, wherein the Bacillus licheniformis is Bacillus licheniformis SD3003 (accession number: FERM BP-5801). 7. Wherein the enzyme having a polyphenol oxidizing action is an enzyme obtained by culturing a fungus of the genus Myrocesium,
The wood treatment method according to any one of the above. 8. 8. The method for treating wood according to the item 7, wherein the fungus of the genus Myrocesium is Myrocesium vercularia or Myrocesium loridum. 9. Myrothecium verrucaria SD3001 (accession number)
FERM BP-5520) or Mirocesium Loridum ( Myroth
ecium roridum ) SD3002 (Accession No. FERM BP-552)
The wood treatment method according to the above item 8, which is 3). 10. 10. The wood treatment method according to any one of 1 to 9, wherein the phenolic compound is lignin or a lignin derivative. 11. The wood treatment method according to the above item 10, wherein the lignin derivative is lignin sulfonic acid. 12. A wood treatment method comprising impregnating wood with an enzyme having a polyphenol oxidizing action in an alkaline pH range and polymerizing a phenolic compound or an aromatic amine compound present in wood with the enzyme. 13. An enzyme having a polyphenol oxidizing action, a phenolic compound or an aromatic amine compound, and one or more heat curing agents selected from quinone compounds, unsaturated fatty acids, unsaturated alcohols and unsaturated alkyl compounds,
A wood treatment method comprising impregnating wood in the H range and polymerizing a phenolic compound or an aromatic amine compound in the wood. 14． An enzyme having a polyphenol oxidizing action, a phenolic compound or an aromatic amine compound, and an antibacterial compound, an antiviral compound, a biological repellent compound, a biocidal compound, and a biologically repellent metal ion selected from a biologically repellent metal ion. A wood treatment method comprising impregnating wood in a pH range and polymerizing a phenolic compound or an aromatic amine compound in the wood.

Hereinafter, the present invention will be described in detail. [Polyphenol oxidase] The enzyme used for the purpose of the present invention may be any enzyme having a polyphenol oxidizing action at alkaline pH. Examples of such enzymes include polyphenol oxidases such as catechol oxidase, laccase, polyphenol oxidase, ascorbate oxidase, or bilirubin oxidase, produced by microorganisms, such as fungi or bacteria, or produced by plants. In addition to these enzymes, any enzyme protein having a polyphenol oxidizing action at an alkaline pH can be used in the present invention.

As the enzyme having a polyphenol oxidizing action in the alkaline pH range used in the present invention, in order to carry out an efficient polymerization reaction, the optimum reaction pH of the polyphenol oxidizing reaction is pH 7.5 or higher. Is desirable. Specifically, it is desirable that such an enzyme has an optimum reaction pH on the alkaline side of pH 7.5 or more in activity measurement using syringaldazine described later.

Examples of microorganisms producing the enzymes used for the purpose of the present invention include the following. As fungi, Aspergillus ( Aspergillus ), Botrytis ( Botryti ) belonging to the subphylum Subuterus ( Deuteromycotina )
s ), Myrothecium , Penicillium ( Pe )
nicillium ), Pestallotia ( Pestalotia ), Rhizoctonia ( Rhizoctonia ), Trichoderma ( Tricoderma ),
Preferably Aspergillus nidulans ( Aspergillu
s nidulans ) and Botrytis cinerea ( Botrytis cin)
erea ), Myrothecium roridu
m ), Myrothecium verrulia
caria ), Myrothecium prestoni ( Myrothecium pre
stonii ), Myrothesium leukotricum ( Myrotheciu)
m leucotrichum), Penicillium sclerotinia Oram (Penicillium sclerotiorum), Penicillium Yanchinerumu (Penicillium janthinellum), Pesutarochia -
Palmarum ( Pestalotia palmarum ), Rhizoctonia
Includes strains belonging to Platicola ( Rhizoctonia praticola ), Tricoderma reesei , and Tricoderma viride . Of these, particularly preferred is Myrothecium verrucaria SD3001 (deposited with FERM BP-5520 at the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology) or Myrothesium Loridaum SD3002
( Myrothecium roridum SD3002) (deposited with the National Institute of Advanced Industrial Science and Technology as a FERM BP-5523).

[0014] Other preferred fungi are Basidiomycetes ( Basidi)
omycotina ), Pleurotus ( Pleurotus ),
Lentinus , Schizophyl
lum), Almira Riera (Armillariella), Furamurina (Flammulina), Agarikasu (Agaricus), Coprinus (Coprinus), Phanerochaete (Phanerochaete), Furebia (Phlebia), Renchitesu (Lenzites), Meranoroika (Melanoleuca), Foriota (Pholiota), Sutereumu ( Stereumu ), Polyporus ( Polyporus ), Polyporellus ( Polyporellus ), Microporous ( Microporus )
us), Fomitopushisu (Fomitopsis), Pikunoporasu (Pycnoporus), Trametes (Trametes), Coriolus (Coriolus), Dedareopushisu (Daedaleopsis), Rijidoporasu (Rigidoporus), Fomesu (Fomes), Ganoderma (Ganoderma), Torakideruma (Trachyderma), Himenokaete ( Hymenochaete), Inonotasu (Inonotu
s ), preferably Pleurotus Cornucopie ( Pleur)
otus cornucopiae ), Pleurotus ostreatus ( Pleurotus osteratus ), Lentina eddes ( L
entinus edodes , schizophyllam commune ( Schizo
phyllum commune , Armillariella melea
riella mellea , Flammulina Bertipes
ina velutipes , Agaricus Bisporus
bisporus ), Coprinus comatu
s ), Coprinus cinereus ,
Coprinus congregatu
s ), Phanerochae
te chrysosporium ), Phlebia radiata ( Phlebia)
radiata), Renchitesu-Betsurina (Lenzites betulin
a ), Melanoleuca verrucipes
cipes), Foriota-nameko (Pholiota nameko), Sutereumu-Hirusutamu (Stereumu hirsutum), Polyporus, Sukuamosasu (Polyporus squamosus), Poriporerusu badius (Polyporellus badius), Mikuroporasu Hula Berri folder Miss (Microporus flabellifor
mis ), Fomitopsis pinico
la), Pikunoporasu-Koshineusu (Pycnoporus coccine
us ), Trametes orienta
lis ), Coriolus versic
olor), Coriolus Hirusutasu (Coriolus hirsutu
s), Dedareopushisu-Torikora (Daedaleopsis tric
olor), Rijidoporasu-Zonarisu (Rigidoporus zonali
s ), Fomes fomentariu
s), Ganoderma lucidum (Ganoderma lucidum), Torakideruma-Tsunodae (Trachyderma tsunodae), Himenokaete-Rubiginosa (Hymenochaete rubiginos
a ), a strain belonging to Inonotus mikadoi .

[0015] Other preferred fungi are Ascomycete subphylum ( Ascomy)
Podosupora belonging to cotina) (Podospora), Neurospora (Neurospora), Monoshiriumu (Monocillium),
Preferably, Podosupora-Anserina (Podospora anse
rina ), Neurospora crass
a ), Monocillium indicum
um ).

[0016] Some preferred bacteria, Azosupiriumu (Azospirillum), preferably Azosupiriumu-Ripoferamu (Azospirillum lipoferum), or, activator flea Seta-less eyes (Actinomycetales), for example, Streptomyces (Streptomyces), preferably Streptomyces Anchibio Ticas ( Streptomyces antibioti
cus ) or Aerobacter , preferably Aerobacter aerogen
es ).

[0017] Other preferred bacteria, Bacillus alkalophilus (Bacillus alcalophilus), Bacillus amyloliquefaciens (Bacillus amyloliquefacien
s), Bacillus brevis (Bacillus brevis), Bacillus Famasu (Bacillus firm us), Bacillus licheniformis (Bacillus licheniformis), Bacillus Natto (Bacillus natto), Bacillus pumilus (Baci
llus pumilus , Bacillus sphaericus ( Bacillus)
sphaericus), Bacillus subtilis (Bacillussubti
lis ), preferably Bacillus licheniformis ( Bacillu
s licheniformis ), Bacillus natus ( Bacillus na)
tto ).

Some preferred plants containing the enzymes used in the present invention include akeshi ( Acerpseudoplatanu).
m ), Yam tree ( Dioscorea ), Okra ( Abelmoschu )
s ), including guava ( Psidium ), sunflower ( Helianthus ), potato, apple, pumpkin, cucumber, wheat, alfalfa, and the like.

[Preparation of enzyme] The enzyme used in the present invention comprises:
In addition to being obtained by culturing the microorganisms described above, for example, strains belonging to fungi or bacteria and mutants thereof, it can also be prepared using genetically engineered bacteria. That is, a DNA sequence encoding the enzyme protein and an appropriate promoter having an enzyme expression function in a host organism, and an operator;
A host cell transformed by using an expression vector inserted into a DNA vector having a replication origin for replicating the vector in a host organism together with a terminator DNA sequence, or a DNA sequence encoding the enzyme protein and a host organism A host cell transformed by incorporating the DNA into a host cell DNA together with an appropriate promoter having an enzyme expression function, an operator, and a terminator DNA sequence is cultured under conditions capable of expressing the enzyme protein. Is also produced by a method of recovering from the medium.

[0020] DN encoding the enzyme protein according to the present invention
In order to obtain the A fragment, an oligonucleotide synthesized based on the amino acid sequence of the enzyme protein of the present invention is obtained by using a cDNA or a genomic library from a microorganism belonging to the aforementioned microorganism, for example, a fungus or a strain belonging to a bacterium, as a separation source. Whether to identify the target DNA fragment as a probe, or to select a clone that expresses the activity as an oxidase,
Alternatively, it can be performed by a conventional method such as selecting a clone that produces a protein that reacts with an antibody against the enzyme protein.

The enzyme protein according to the present invention can also be prepared by extracting from the above-mentioned plant-derived seeds, fruits, leaves and the like. For culturing strains belonging to fungi or bacteria and mutants thereof for obtaining the enzyme protein according to 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. In the case of culture, C
It is desirable to add u ²⁺ ion as a metal salt at a concentration of 0.001 mM to 10 mM, preferably 0.01 mM to 1 mM.

When the polyphenol oxidase of the present invention is secreted outside the fungal or bacterial cells, it 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 by, for example, ion exchange chromatography. Further, membrane concentration using an ultrafiltration membrane is also effective. When the enzyme protein accumulates in fungal or bacterial cells or is present in plant tissues, it can be recovered from the cell tissues or plant tissues by a well-known method. This recovery procedure involves mechanical disruption of tissue by homogenization,
It includes a series of procedures of separating and extracting an enzyme protein solution by centrifugation, filtration, or membrane separation, and performing, for example, ion exchange chromatography. Further, membrane concentration using an ultrafiltration membrane is also effective.

[Activity Measurement Method] In the present invention, the measurement of the polyphenol oxidizing activity of the enzyme protein having a polyphenol oxidizing effect was carried out at 20 ° C. at 20 ppm syringaldazine and 100 mM T
The reaction was performed at an optimum reaction pH in an aqueous solution containing a ris-HCl buffer or a potassium phosphate buffer, and the measurement was performed by measuring the absorbance at 525 nm. The activity of oxidizing 1 μmol of syringaldazine per minute was defined as 1 unit (hereinafter abbreviated as U).

[Polymerization reaction method and use thereof]
A phenolic compound of increased molecular weight by the method or
Phenolic compounds in the production of aromatic amine compounds
The concentration of a substance or an aromatic amine compound is 0.01 to 90%, preferably
Preferably it is 1 to 80%. The reaction temperature is 0 to 15
0 ° C., preferably 0-100 ° C. In addition, the reaction
The pH is between 7.0 and 12, preferably between 7.5 and 10. Also,
The enzyme activity concentration to be used is 1 to 10,000 U / liter, preferably
Or 10 to 2000 U / liter. Enzyme activity concentration
It is desirable to adjust according to the purpose. That is,
Want to achieve rapid polymerization and gelation or solidification
In this case, the reaction may be performed at a high activity concentration. On the other hand, low
If the reaction is performed at an active concentration, a mild polymerization reaction proceeds
To obtain a more uniform polymer solution as a liquid substance
When the reaction is continued, a gentle gelation reaction
Proceeds throughout the reaction. To a suitable degree of polymerization
The reaction was stopped at this point by using NaOH, NH _Three, Na_TwoCO
_Three, CaCO_ThreeAddition of alkali or alkali salt such as salt
Addition of acids such as acid, sulfuric acid and nitric acid, addition of known enzyme inhibitors
Or heat treatment at 100 ° C for 15 minutes.
Can be implemented.

The gelled phenolic compound or aromatic amine compound can be dissolved again by heating at 50 to 230 ° C. if desired. Such heat solubility is a useful property when used in applications such as dispersants, adhesives, and paints. Further, by adding hot water or the like after the heat dissolution and dispersing and dissolving the same, it is possible to obtain a phenolic compound or an aromatic amine compound having a very high molecular weight as a solution.

For the purpose of promoting curing by heating,
It is also possible to add polyols such as furfuryl alcohol and sugar. In addition, for the purpose of containing a biologically active substance in a polymer compound and obtaining a substance having an immobilized substance of the physiologically active substance or a sustained release of the physiologically active substance, an antibacterial compound, an antiviral compound, a biological repellent compound, Perform the polymerization reaction in the presence of an insecticide compound or metal ion, or add an antibacterial compound, antiviral compound, biological repellent compound, insecticidal compound or metal ion after the polymerization reaction. Is also possible. Antibacterial compounds, antiviral compounds, biological repellent compounds, insecticidal compounds or metal ions used for this purpose include:
Many conventionally known substances can be used.

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. Enable application. Also,
In the case of producing a large amount of a polymerized product, 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 allow the reaction to proceed simultaneously.

[Phenolic compound or aromatic amine compound] As the phenolic compound or aromatic amine compound to be polymerized in the present invention, any compound can be used as long as the enzyme used in the present invention can be oxidized. It is possible. Specific examples of such phenolic compounds or aromatic amine compounds include lignin, ligninsulfonic acid, humic acid, nitrohumic acid, tannin,
Catechin, gallic acid, urushiol, hesperidin, chlorogenic acid, hinokitiol, pyrocatechol, hydroquinone, t-butylhydroquinone, phenylhydroquinone, trimethylhydroquinone, ethyl 3,4
-Dihydroxycinnamic acid, pyrogallol, lauryl gallate, octyl gallate, syringic acid, ferulic acid, vanillin, o-vanillin, vanilla acid, vanillyl alcohol, ascorbic acid, 1,2-dihydroxynaphthalene, 2,3-dihydroxynaphthalene, 6,7-dihydroxy-2-naphthalenesulfonic acid, anthralobin, alizarin, quinizarin, o-phenylenediamine, p-phenylenediamine, 3,4-diaminobenzophenone, o-anisidine, p-anisidine, o-aminophenol, p -Aminophenol, 1,2-diaminoanthraquinone and 1,4-diaminoanthraquinone.

In addition to these compounds, any substance capable of oxidizing the enzyme used in the present invention can be used as a raw material for a polymerized product or as a catalyst for a polymerizing reaction. Examples of such compounds include ABTS (2,2'-azobis (3-ethylbenzothiazoline-6-sulfonic acid)), bilirubin, isoascorbic acid, quercetin, rutin, guaiacol, 4-methoxyphenol, biphenol, , 4'-ethylenedianiline, methylhydroquinone, 1-hydroxybenzotriazole, 6-hydroxy-2,4,5-triaminopyrimidine, 4,5,6-triaminopyrimidine, 2,3-dihydroxypyridazine, 3, 6-dihydroxypyridazine, 2,3-dihydroxypyridine, 4-hydroxy-
3-methoxybenzoic acid, methyl 4-hydroxy-3-methoxybenzoic acid, 4,5-diamino-6-hydroxy-
2-mercaptopyrimidine, 2,3-diaminopyridine, 2,5-dihydroxy-1,4-benzoquinone,
2,5-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,4-dihydroxy-3-cyclobutene-
1,2-dione, 3- (3,4-dihydroxyphenyl) -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, 3,4-dimethoxybenzyl alcohol, 3,4-dimethoxyphenethylamine, (3,4
-Dimethoxyphenyl) acetic acid, (3,4-dimethoxyphenyl) acetonitrile, 4-aryl-2-methoxyphenol, 2-methoxy-4-propphenylphenol, 2-methoxy-5-methylaniline, 2-methoxy-5 Nitroaniline, 4-methoxy-2-nitroaniline, 3-methoxysalicylic acid, 3-methylcatechol, 4-methylcatechol, methylgallate, propylgallate, 3,4,5-trimethoxyaniline, 3,
4,5-trimethoxyphenol, tropolone, purprogalin, salicylaldoxime, 3-amino-5
6,7,8-tetrahydro-2-naphthol, 1,5-
Dihydroxynaphthalene, 3,5-dihydroxy-2-
Naphthoic acid, 4-hydroxy-1-naphthalenesulfonic acid, purpurin, 2,3-dihydro-9,10-dihydroxy-1,4-anthracenedione, and various azo dyes. Further, for the purpose of adjusting the physical properties of the polymerized product, a plurality of these phenolic compounds or aromatic amine compounds can be used in combination.

In producing a high molecular phenolic compound or an aromatic amine compound according to the present invention, a quinone compound which is polymerized by a similar reaction route may be used. Examples of such quinone compounds are
Anthraquinone-2-sulfonic acid, anthraquinone-
1,5-disulfonic acid, anthraquinone-2,6-disulfonic acid, anthraquinone-2-carboxylic acid, 1-aminoanthraquinone, 2-aminoanthraquinone, anthralphine, aminonaphthoquinone, 1,8-dihydroxyanthraquinone , Camphoquinone, dehydroascorbic acid, 2-hydroxy-1,4-naphthoquinone, isatin, 5-nitroisatin, and various anthraquinone dyes. In addition, unsaturated fatty acids such as oleic acid and linoleic acid, or unsaturated alcohols such as oleyl alcohol, and an autoxidizable substance such as unsaturated alkyl such as squalene coexist with air oxidation and polymerization simultaneously with the enzymatic reaction. It is also possible to do.

Among the high molecular phenolic compounds or aromatic amine compounds prepared according to the present invention, especially lignin, ligninsulfonic acid, humic acid, nitrohumic acid, tannin, catechin, gallic acid, urushiol, hesperidin, hinokitiol, etc. The polymerized product of natural products or natural product derivatives is highly useful because of its high safety to the environment and the human body.Taking advantage of the properties of the polymer compound, thickeners, stabilizers, flocculants, emulsifiers, Dispersant, water retention agent, antioxidant, adhesive, concrete admixture, dye, paint, oil recovery agent, soil modifier, seed spraying topsoil stabilizer, deodorant, deodorant, pesticide spreading agent, feed A binder,
It can be used in various application fields such as a bactericide, an antibacterial agent, a virus infection inhibitor, a biofouling inhibitor, a biorepellent, an insecticide, a cataplasm, an ink base or a wood treatment agent. In these applications, various additives commonly used in each field can be used in combination.

Further, in these fields of use, the production method disclosed in the present invention polymerizes a natural or unnatural phenolic compound or aromatic amine compound under mild reaction conditions, and provides viscosity, adhesion, and water retention. By adjusting physical properties such as water solubility, water resistance, elasticity, and strength, and physiological functions, it is possible to develop a more highly functional application as a polymer compound.

Further, the enzyme having a polyphenol oxidizing effect is acted on wastewater containing a phenolic compound or an aromatic amine compound in an alkaline pH range according to the present invention, whereby the phenolic compound or the aromatic amine compound in the wastewater can be highly purified. A wastewater treatment method is also possible in which the phenolic compound or the aromatic amine compound that has been polymerized is separated and removed from the wastewater so that the phenolic compound or the aromatic amine compound that has been polymerized can be easily concentrated. Industrial fields and reaction substrates for which such use is particularly useful include lignin or lignin derivatives in the paper pulp field and azo and anthraquinone dyes in the coloring and dyeing fields. After such a polymerization reaction, coagulation sedimentation treatment by addition of a coagulant, activated carbon treatment, and filtration treatment enable efficient concentration and separation / removal of phenolic compounds or aromatic amine compounds in wastewater from wastewater. It is possible.

Further, according to the present invention, a deoxygenation method or production of an oxygen scavenger utilizing the action of an enzyme having a polyphenol oxidizing action on a phenolic compound or an aromatic amine compound in an alkaline pH range to consume dissolved oxygen. Is also possible. In such deoxygenation methods and oxygen absorbers, many natural or non-natural phenolic compounds or aromatic amine compounds can be used, and the dissolved oxygen concentration can be rapidly reduced, which is extremely useful.

Further, according to the present invention, wood is impregnated with an enzyme having a polyphenol oxidizing action together with a phenolic compound or an aromatic amine compound, and the phenolic compound or the aromatic amine compound, and also lignin already contained in the wood. Polymerization of polyphenol compounds such as in wood improves workability in the drying process after wood impregnation, improves wood strength reduced by lignin decomposition by wood steaming or high-temperature steam injection, and improves drying. Or improvement of action to prevent cracking of wood during freezing,
It is possible to suppress the growth of microorganisms by maintaining and improving the anaerobic environment in wood.

According to the present invention, a dye or a dye precursor capable of acting on a polyphenol oxidase and a polyphenol oxidase are allowed to act on wood to produce a coloring substance in the wood, or the coloring substance and the Since the polyphenol compound such as lignin contained therein can be made into a composite polymer in wood, the wood can be dyed and colored more strongly. In the above wood dyeing / coloring treatment, it is known that many polyphenol oxidases bleach lignin, which is a coloring substance in wood, and the wood dyeing / coloring treatment of the present invention involves enzymatic bleaching and coloring. Since dyeing and coloring can be performed simultaneously, the process is shortened and the color tone is improved, which is extremely useful.

In addition, an enzyme having a polyphenol oxidizing action according to the present invention is added to concrete together with a phenolic compound or an aromatic amine compound, and the phenolic compound or the aromatic amine compound is polymerized in the concrete, thereby producing a slump. It is possible to improve the loss, increase the concrete strength, and control the rust of the reinforcing steel by lowering the oxygen concentration in the concrete.

[0038]

The present invention will be described more specifically below with reference to typical examples. However, these are merely examples, and the present invention is not limited to only these. In the following example, the molecular weight analysis of a polymerized phenolic compound or aromatic amine compound was performed using a 50 mM potassium phosphate buffer (pH 7.0) as an eluent.
Alternatively, a 0.1 mM sodium sulfate aqueous solution, Shodex RI (differential refractive index detector) is used as a detector, and Shodex PROTEIN KW-802.5 (dual) or Shodex PROTEIN KW802.5 and Shodex OHpak SB-804H are used as columns.
Performed by HPLC using a Q tie.

Example 1: Culture, crude purification, concentration A 500 ml flask was used as a culture device, and 0.134% N
_{a 2 HPO 4 · 12H 2 O} , 0.03% KH 2 PO 4, 1% maltose, 1% peptone, 0.1% yeast extract, 0.05% Mg
_{SO 4 · 7H 2 O, 0.1mM} CuSO 4, 1mM MnC
l ₂ , 20 mM in 100 ml of medium containing ₂ mM CaCl ₂
% Na ₂ CO ₃ to adjust the pH to 7.8, and Bacillus licheniformis SD3
003 (Accession No. FERM BP-5801) at 50 ° C, 1
After the shaking culture for 6 hours, the culture temperature was lowered to 35 ° C., and the culture was further performed for 3 days. After the cultivation, a culture broth was removed by centrifugation at 4 ° C. This is further purified,
Ammonium sulfate fractionation was effective for concentration, and most of the polyphenol oxidase activity could be recovered as a precipitate at a saturated ammonium sulfate concentration of 20 to 60%. The obtained ammonium sulfate precipitate was dissolved in a 10 mM bis-tris-HCl buffer solution (p
H7.0), and an ultrafiltration membrane was used for further purification and concentration to obtain a crudely purified concentrated aqueous solution (0.8 U / ml) in the range of 10,000 to 100,000 in molecular weight.

Example 2: Culture and concentration 0.5% glucose and 0.1% NaNO ₃ , 1.34% Na ₂ HP
O ₄ 12H ₂ O, 0.3% KH ₂ PO ₄ , 0.1% NaCl, 0.2
% Peptone, 20 ppm yeast extract, 0.01% MgSO ₄
· 7H ₂ O, 0.1mM CuSO consists ₄ 3 liters of medium Myrothecium, the culture tank including those with 8 to pH by the addition of 10% NaOH to Verukaria (Myrothecium
verrucaria ) SD3001 (Accession No. FERM BP-5520) and inoculated at 28 ° C. for 3 days with shaking. After culturing, culture broth 2.5 centrifuged at 4 ° C.
I got a liter. Next, a part of the culture broth was concentrated as a fraction having a molecular weight of 10,000 or more by a minitan ultrafiltration system (manufactured by Millipore) using a minitan filter packet (CAT. NO .: PTGC0MP04, manufactured by Millipore). . This was further dialyzed against 200 ppm NH ₄ HCO ₃ and then freeze-dried to obtain a crude product as a freeze-dried product. The polyphenol oxidase activity of the freeze-dried product was 15 U / mg. In addition, the aqueous solution of this freeze-dried product showed an absorption maximum at around 600 nm which is specific to the copper-containing protein.

Example 3: Culture and concentration 0.5% glucose and 0.1% NaNO ₃ , 1.34% Na ₂ HP
O ₄ .12H ₂ O, 0.3% KH ₂ PO ₄ , 0.1% NaCl, 0.1%
2% peptone, 20 ppm yeast extract, 0.01% MgSO ₄
· 7H ₂ O, the culture tank including those with 8 to pH by addition of 3 liters medium of 10% NaOH consisting 0.1 mM CuSO _4, Myrothecium, Roridamu (Myrothecium ro
ridum ) SD3002 (Accession No. FERM BP-5523), and shaking culture was performed at 28 ° C. for 3 days. After culture, 4
2.5 liters of the culture broth that had been sterilized was obtained by centrifugation at ° C. Next, a part of this culture broth was
It was concentrated as a fraction having a molecular weight of 10,000 or more by a minitan ultrafiltration system (manufactured by Millipore) using a filter packet (CAT. NO .: PTGC0MP04, manufactured by Millipore). This was further dialyzed against 200 ppm NH ₄ HCO ₃ and then freeze-dried to obtain a crude product as a freeze-dried product. The polyphenol oxidase activity of the lyophilized product was 10 U / mg. In addition, the aqueous solution of this freeze-dried product showed an absorption maximum at around 600 nm which is specific to the copper-containing protein.

Example 4 Polymerization Reaction Using the roughly purified concentrated aqueous solution described in Example 1, the following [4-
Polymerization reactions of [1] to [4-3] were performed using commercially available polyphenol oxidase (obtained from Takara).

[4-1]: Lignin sulfonic acid sodium salt (Aldrich Chemical Company)
emical Company, Inc.) 20% (W /
V), 1 m of a reaction solution containing a roughly purified concentrated aqueous solution as a polyphenol oxidase at an active concentration of 300 U / liter
1 in a glass test tube at a reaction temperature of 70 ° C.
The reaction was carried out by shaking at 100 rpm. The pH of the reaction solution was adjusted to 7.5 with a small amount of sulfuric acid. After the reaction starts,
Immediately after 6 hours, the color tone of the reaction solution became deep, and after 6 hours, remarkable progress of polymerization was observed, and after 20 hours, most of the reaction solution was solidified.

[4-2]: When the reaction was carried out while changing the activity concentration of polyphenol oxidase to 60 U / liter, the whole reaction liquid exhibited a highly viscous liquid 20 hours after the start of the reaction, The molecular weight increased with the increase, and after continued reaction for another 20 hours, partial solidification was observed. In addition, the sample for molecular weight analysis was prepared by extracting a part of the reaction solution, performing heat treatment at about 100 ° C. for 15 minutes in a water bath, and stopping the reaction.

[4-3] Lignin (alkali) (obtained from nacalai tesque) at a concentration of 20% (W / V) in place of lignin sulfonic acid, and an activity concentration of polyphenol oxidase of 300 U / liter When the reaction was carried out at pH 7.5, the entire reaction liquid was a highly viscous liquid 24 hours after the start of the reaction.

[4-4]: Lignin sulfonic acid sodium salt is 20% (W / V), and 300 U of commercially available polyphenol oxidase is used as polyphenol oxidase.
A reaction solution containing 100 ml / liter of active concentration was prepared, and the reaction temperature was set to 25 in a 500 ml flask.
The reaction was carried out by shaking at 100 ° C. and 100 rpm. The commercially available polyphenol oxidase used here was shown to be an acidic enzyme having an optimum reaction pH of pH 6 to 7 by activity measurement using syringaldazine, so that the pH of the reaction solution in the polymerization reaction was It 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 reaction solution to solidify most of the reaction solution.

Example 5 Polymerization Reaction The lyophilized product described in Example 2 was used to carry out the following polymerization reactions [5-1] to [5-3].

[5-1]: 100 ml of a reaction solution containing 20% (W / V) of ligninsulfonic acid sodium salt and 20 ppm (300 U / liter) of a lyophilized product as polyphenol oxidase was prepared, and 500 ml of the reaction solution was prepared. In the volumetric flask, the reaction was carried out by shaking at a reaction temperature of 25 ° C. and 100 rpm. The pH of the reaction solution was adjusted to 9 using NaOH. Immediately after the start of the reaction, the color tone of the reaction solution became dark, remarkable progress of polymerization was observed after 3 hours, and most of the reaction solution was solidified after 10 hours. When the reaction was carried out with the added amount of polyphenol oxidase changed to 4 ppm, the whole reaction solution became highly viscous in 20 hours after the start of the reaction, and was partially solidified after the reaction was continued for 20 hours. Was observed. For the sample for molecular weight analysis, a part of the reaction solution was withdrawn and placed in a water bath.
It was prepared by performing a heat treatment at 0 ° C. for 5 minutes to stop the reaction.

[5-2]: Lignin (alkali) was used at a concentration of 20% (W / V) instead of ligninsulfonic acid, the activity concentration of polyphenol oxidase was 300 U / liter, pH 9 and reaction temperature was 25. The reaction was performed at ° C. Immediately after the start of the reaction, the color tone of the reaction solution became dark, the polymerization reaction proceeded, and after 24 hours, most of the reaction solution was solidified.

[5-3]: The ligninsulfonic acid sodium salt was adjusted to 20% (W / V) at an activity concentration of 300 U / liter of polyphenol oxidase, and the pH of the reaction solution was reduced to 7 using a small amount of sulfuric acid. The temperature was adjusted and the reaction was carried out at a reaction temperature of 25 ° C. Immediately after the start of the reaction, the color tone of the reaction solution became dark, but no solidification of the reaction solution was observed.

Example 6: Polymerization reaction Using the freeze-dried product described in Example 3, a polymerization reaction of the following [6-1] to [6-2] was performed.

[6-1]: 100 ml of a reaction solution containing 20% (W / V) of ligninsulfonic acid sodium salt and 30 ppm (300 U / liter) of a lyophilized product as polyphenol oxidase was prepared, and 500 ml of the reaction solution was prepared. In the volumetric flask, the reaction was carried out by shaking at a reaction temperature of 25 ° C. and 100 rpm. The pH of the reaction solution was adjusted to 9 using NaOH. Immediately after the start of the reaction, the color tone of the reaction solution became dark, remarkable progress of polymerization was observed after 3 hours, and most of the reaction solution was solidified after 10 hours.

[6-2]: Lignin (alkali) is used at a concentration of 20% (W / V) instead of lignin sulfonic acid, the activity concentration of polyphenol oxidase at 300 U / liter, pH 9, reaction temperature 25 The reaction was performed at ° C. Immediately after the start of the reaction, the color tone of the reaction solution became dark,
After the time, most of the reaction solution was solidified.

Example 7: Polymerization reaction Lignin sulfonic acid sodium salt was 20% (W / V)
Commercially available bilirubin oxidase (lyophilized product) as polyphenol oxidase (obtained from Sigma)
Of a reaction solution containing 300 U / liter
was prepared and shaken at a reaction temperature of 25 ° C. and 100 rpm in a 500-ml flask to carry out the reaction.
Since the commercially available bilirubin oxidase used here was an enzyme having an optimum reaction pH of pH 8 to 9 in activity measurement using syringaldazine, the pH of the reaction solution in the polymerization reaction was not measured. The adjustment was performed at pH 8.5 as it was. Immediately after the start of the reaction, the color tone of the reaction solution became dark, and after 8 hours, remarkable progress of polymerization was observed.
After 4 hours, most of the reaction solution had solidified. In addition, lignin (alkali) is replaced by 20% instead of ligninsulfonic acid.
(W / V), and the reaction was carried out at pH 8.5. As a result, most of the reaction solution was solidified 50 hours after the start of the reaction.

Example 8: Antibacterial activity Solid (gel-like) ligninsulfonic acid obtained by performing the same reaction as the polymerization reaction described in [5-1] of Example 5 was further reduced into small pieces (10 mm × A slice of 10 mm x 3 mm) was placed in the center of an L agar plate, and further Aspergillus oryzae AHU7134 ( Aspergil
lus oryzae AHU7134) was added to the entire surface of the medium of the plate, and cultured at 28 ° C for 4 days. As a result, the growth of ligninsulfonic acid was inhibited in the upper part of the small piece and within a range of about 2 mm around the ligninsulfonic acid. It has been shown to be useful as an agent.

Example 9: To an aqueous solution of ligninsulfonic acid sodium salt having an antibacterial property of 20% (W / V), hinokitiol (obtained from Tokyo Chemical Industry Co., Ltd.) at a concentration of 200 ppm was added and heated at 90 ° C. To dissolve the hinokitiol, and suspended the hinokitiol with a vortex mixer, followed by cooling to 25 ° C. The lyophilized product described in Example 2 was used as a polyphenol oxidase in 30 parts of the raw material for the polymerization reaction thus obtained.
0 U / liter at an active concentration of 25 ° C.
Polymerization reaction was performed at pH 9 to obtain hinokitiol-containing solid (gel) ligninsulfonic acid. The solid material was sliced into small pieces (10 mm × 10 mm × 3 mm), placed in the center of an L agar plate, and further subjected to Aspergillus oryzae AHU1344.
U7134) spores are added to the entire medium surface of the plate and
After culturing for 4 days, the growth of the lignin sulfonic acid was inhibited in the upper part of the small piece and in a range of about 10 mm around the small piece, indicating that the lignin sulfonic acid is useful as an antibacterial agent and an antibacterial substance holding agent. Also, (+)-catechin.
H ₂ O (obtained from Sigma) was added at a concentration of 5000 ppm, and further as a polyphenol oxidase in Example 2
The freeze-dried product described above was added at an active concentration of 300 U / liter, and a polymerization reaction was carried out at a reaction temperature of 25 ° C. and pH 9 to obtain a catechin-containing solid (gel-like) ligninsulfonic acid.
When the antibacterial property of this solid was examined in the same manner as in the above example, it was found that the growth of bacteria was inhibited in the upper part of the lignin sulfonic acid piece and in a range of about 8 mm around the ligninsulfonic acid. It was shown that there is.

Example 10: Wood treatment (Example 1 of the original application)
4) A reaction solution containing the lyophilized product described in Example 2 at a concentration of 4 ppm as a polyphenol oxidase was prepared, and the reaction was carried out in the same manner as in the polymerization reaction described in [5-1] of Example 5. , Water soluble polymerized lignin sulfonic acid (20%
(W / V)). The operation of stopping the polymerization reaction by heat or the like was not performed. This aqueous polymer solution is washed with water
The solution diluted to 0-fold was used as a wood treatment chemical, and after removing the bark, it was treated with a 0.1% Tween 80 aqueous solution at 60 ° C. for 16 hours for a cedar log (raw tree 3 cm in diameter and 20 cm in length). A drug injection process was performed. The injection process is 720mmH
The pressure was 10 g / cm ² and the pressure was 10 kg / cm ² . Wood (two each) injected with lignin sulfonic acid or non-polymerized lignin sulfonic acid or polymerized lignin sulfonic acid was placed on the soil about 20 cm around the termite nest and left for 2 months. Later, when the termite-prevention effect of the chemical treatment was observed, wood treated with ligninsulfonic acid that had not been polymerized showed slight damage by termites, whereas wood that had been treated with polymerized ligninsulfonic acid showed slight damage. No termite damage was observed, and the uniformity (smoothness, gloss) of the wood surface was maintained.

Example 11: Wood treatment (Example 1 of the original application)
5) A 1% aqueous solution of p-phenylenediamine / dihydrochloric acid (obtained from Kanto Chemical Co., Ltd.) was adjusted to pH
9 to which the freeze-dried product described in Example 2 was added as a polyphenol oxidase at a concentration of 0.2 ppm,
Immediately, it was applied to oak board. The coloring reaction proceeded rapidly on the surface and inside (depth to about 3 mm) of the plate to obtain a plate material that was strongly colored blackish brown.

REFERENCE EXAMPLE 1: Concrete modification (Original application implemented)
Example 10) Lignin sulfonic acid sodium salt at 20% (W / V) and anthraquinone-2-sulfonic acid at 1% (W / V),
A reaction solution containing the roughly purified concentrated aqueous solution described in Example 1 as the polyphenol oxidase at an active concentration of 60 U / liter was prepared, and reacted in the same manner as in the polymerization reaction described in [4-2] of Example 4. And the obtained water-soluble polymer was reduced to 1/40 volume (final concentration 0.5% as ligninsulfonic acid).
% Weight), sand rate (S / A) 36% weight, cement 440
When mixed with g / liter, the slump was 5.5 cm and the water reduction was 16.8%. In addition, when the same test as the above example was performed without performing the polymerization reaction, the slump was 5.7 cm, the water reduction rate was 12.5%, and the improvement of the water reduction effect by the polymerization was recognized.

Reference Example 2: Concrete modification (implementation of original application)
Example 11) Lignin sulfonic acid sodium salt at 20% (W / V) and anthraquinone-2-sulfonic acid at 1% (W / V),
A reaction solution containing the freeze-dried product described in Example 2 at a concentration of 4 ppm as a polyphenol oxidase was prepared and reacted in the same manner as in the polymerization reaction described in [5-1] of Example 5 to obtain a reaction solution. 1/40 volume of water-soluble polymer (final concentration 0.5% weight as ligninsulfonic acid), sand ratio (S /
A) When mixed with 36% by weight of cement and 440 g / liter of cement, the slump was 5.6 cm and the water reduction was 16.9%.
In addition, when the same test as in the above example was performed without performing the polymerization reaction, the slump was 5.4 cm and the water reduction rate was 1
It was 2.8%, indicating an improvement in the water reducing effect due to polymerization.

Reference Example 3: Soil modification (Example 12 of the original application) A reaction solution containing the roughly purified concentrated aqueous solution described in Example 1 at an active concentration of 60 U / liter as a polyphenol oxidase was prepared. The reaction was carried out in the same manner as the polymerization reaction described in [4-2] to obtain a water-soluble polymerized ligninsulfonic acid (20% (W / V)), which was diluted twice with water. Prepared. The operation of stopping the polymerization reaction by heat or the like was not performed. 3.0 ml of this polymerized lignin sulfonic acid aqueous solution or 3.0 ml of water was placed in a 50 ml glass beaker containing 40 g of field soil,
The soil was sprayed, incubated at 28 ° C., and the weight loss due to drying was measured. After 60 hours, the weight of the polymerized lignin sulfonic acid solution was reduced by about 6 g, and the weight of the sprayed water was reduced by about 12 g, indicating that the water retention of the polymerized lignin sulfonic acid was improved. Also, the spraying of the polymerized lignin sulfonic acid solution showed an improvement in the hardness of the soil surface, indicating that it was useful as a soil modifier and a seed spraying topsoil stabilizer.

Reference Example 4: Soil modification (Example 13 of the original application) A reaction solution containing the lyophilized product described in Example 2 at a concentration of 4 ppm as a polyphenol oxidase was prepared, and [5-1] of Example 5 was used. The reaction was carried out in the same manner as in the polymerization reaction described in [1], and water-soluble polymerization ligninsulfonic acid (20%
(W / V)), and a two-fold dilution with water was prepared. The operation of stopping the polymerization reaction by heat or the like was not performed. This polymerized lignin sulfonic acid aqueous solution 3.0
ml or 3.0 ml of water was sprayed on the soil surface in a 50 ml glass beaker containing 40 g of field soil, incubated at 28 ° C., and the weight loss due to drying was measured. After 60 hours, the weight of the polymerized lignin sulfonic acid solution was reduced by about 5 g, and the weight of the sprayed water was reduced by about 12 g, indicating that the water retention of the polymerized lignin sulfonic acid was improved. Also, the spraying of the polymerized lignin sulfonic acid solution showed an improvement in the hardness of the soil surface, indicating that it was useful as a soil modifier and a seed spraying topsoil stabilizer.

Reference Example 5 Wastewater Treatment (Example 16 of the Original Application) As a model of wastewater containing a phenolic compound or an aromatic amine compound, a 10 mM p-phenylenediamine / dihydrochloric acid aqueous solution was used. To this aqueous solution was added 2 μl of the lyophilized product described in Example 2 as a polyphenol oxidase.
Prepare 100 ml of reaction solution added at a concentration of pm
In a 500 ml flask under the conditions of 8, a reaction temperature of 2
Polymerization reaction was carried out by shaking at 5 ° C. and 60 rpm. Immediately after the start of the reaction, coloring of the reaction solution started, and one hour later, remarkable coloring due to progress of oxidation and polymerization and aggregation of the polymer compound were observed. This reaction solution was passed through a simple column containing a powdered cellulose, KC Floc (obtained from Nippon Paper Industries Co., Ltd.), as a filter medium in a volume of about 40 cm ³ , and a large amount of a polymer compound derived from p-phenylenediamine was obtained. The part could be filtered off from the aqueous solution. As the polyphenol oxidase, the crude purified concentrated aqueous solution described in Example 1 was used at an active concentration of 30 U / liter instead of the lyophilized product 2 ppm described in Example 2, and p-phenylene was used in the same manner as in the above example. When the diamine was treated, most of the high molecular compound derived from p-phenylenediamine could be similarly filtered from the aqueous solution.

Reference Example 6: Oxygen scavenger (Example 17 of the original application) A freeze-dried product described in Example 2 at pH 9.0 and a temperature of 25 ° C. using 50 mM L-ascorbic acid / Na as a phenolic compound. At a concentration of 4 ppm,
When the oxygen consumption rate was measured with a manometer, the dissolved oxygen concentration one hour after the start of the reaction was reduced to 0.05% at the start of the reaction, indicating a high deoxygenation ability. The oxygen consumption rate was measured in the same manner as in the above Example except that the crude purified concentrated aqueous solution described in Example 1 was used at an active concentration of 30 U / liter instead of 4 ppm of the lyophilized product described in Example 2. The dissolved oxygen concentration one hour after the start of the reaction was reduced to 0.2% at the start of the reaction, indicating a similarly high deoxygenation ability.

[0065]

According to the present invention, an efficient polymerization reaction of a phenolic compound or an aromatic amine compound is achieved, and a thickener, a stabilizer, a coagulant, an emulsifier, a Agent, water retention agent, antioxidant, adhesive, concrete admixture, dye, paint, oil recovery agent, soil modifier, seed spraying topsoil stabilizer, deodorant, deodorant, pesticide spreading agent, feed A binder, a bactericide, an antibacterial agent, a virus infection inhibitor, a biofouling inhibitor, a biorepellent, an insecticide, a poultice, an ink base or a wood treating agent are efficiently provided.

Further, by utilizing the present invention, a thickener, a stabilizer, a flocculant, an emulsifier, a dispersant, a water retention agent, an antioxidant, and a step of polymerizing a phenolic compound or an aromatic amine compound are provided. Agents, adhesives, concrete admixtures, dyes, paints, oil recovery agents, soil modifiers, seed spray topsoil stabilizers, deodorants, deodorants, pesticide spreading agents, feed binders, fungicides, antibacterial agents And a method for efficiently producing a virus infection inhibitor, a biofouling inhibitor, a biorepellent, an insecticide, a poultice, an ink base or a wood treating agent.

Further, according to the present invention, there are provided a wastewater treatment method, a deoxygenation method, a wood treatment method, a concrete treatment method and a soil treatment method utilizing a polymerization reaction of a phenolic compound or an aromatic amine compound. . In addition, Bacillus liceniformis used in the present invention.
heniformis ) SD3003, Myrothecium verrucaria SD3001, Myrothecium roridum SD3002
Is particularly useful for producing the polymerized product of the present invention.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) B27K 5/00 B27K 5/00 Z C12S 3/02 C12S 3/02 (72) Inventor Ritsuko Ohno Tokyo Metropolitan Port 1-7-15 Ward Shiba Park Shoko Tsusho Co., Ltd. Shodex Service Center F term (reference) 2B230 AA16 BA01 CB10 EA23 EB02 4H011 AA02 AC03 BA06 BB05 BB19 BC18 DA13 DA17 DD03 DH10

Claims (14)

[Claims] The present invention is characterized in that an enzyme having a polyphenol oxidizing action is impregnated in wood together with a phenolic compound or an aromatic amine compound in an alkaline pH range, and the phenolic compound or aromatic amine compound is polymerized in the wood. And wood processing method. 2. The wood treatment method according to claim 1, wherein the wood is polymerized in an alkaline region having a pH of 8 or more. 3. The wood treatment method according to claim 1, wherein catechol oxidase, laccase, polyphenol oxidase, ascorbate oxidase, or bilirubin oxidase is used alone or in combination as an enzyme having a polyphenol oxidizing action. 4. The wood treatment method according to claim 1, wherein the enzyme having a polyphenol oxidizing action is an enzyme obtained by culturing a bacterium belonging to the genus Bacillus. 5. The bacterium belonging to the genus Bacillus, wherein the bacterium belongs to the genus Bacillus.
5. The method for treating wood according to claim 4, wherein the method is licheniformis or Bacillus natto. 6. Bacillus licheniformis SD30, wherein Bacillus licheniformis is Bacillus licheniformis SD30.
No. 03 (Accession number FERM BP-5801). 7. The method for treating wood according to claim 1, wherein the enzyme having a polyphenol oxidizing action is an enzyme obtained by culturing a fungus of the genus Myrocesium. 8. The microorganism of the genus Myrosesium,
The wood treatment method according to claim 7, which is Vercaria or Milosesium loridum. 9. The fungus of the genus Myrocesium is Myrocesium.
Verkalia ( Myrothecium verrucaria ) SD3001
(Accession number FERM BP-5520) or Myrothecium roridum SD3002 (Accession number FER)
The wood treatment method according to claim 8, which is MBP-5523). 10. The method for treating wood according to claim 1, wherein the phenolic compound is lignin or a lignin derivative. 11. The method according to claim 10, wherein the lignin derivative is ligninsulfonic acid. 12. A method for treating wood, comprising impregnating wood with an enzyme having a polyphenol oxidizing action in an alkaline pH range, and polymerizing a phenolic compound or an aromatic amine compound present in wood with the enzyme. . 13. An enzyme having a polyphenol oxidizing effect, a phenolic compound or an aromatic amine compound, and one or more heat curing agents selected from quinone compounds, unsaturated fatty acids, unsaturated alcohols and unsaturated alkyl compounds, in an alkaline pH range. The wood treatment method according to claim 1, wherein the phenolic compound or the aromatic amine compound is polymerized in the wood by impregnating the wood. 14. A bioactive substrate selected from an enzyme having a polyphenol oxidizing action, a phenolic compound or an aromatic amine compound, and an antibacterial compound, an antiviral compound, a biological repellent compound, an insecticidal compound, and a biological repellent metal ion. A wood treatment method comprising impregnating wood with at least one in an alkaline pH range and polymerizing a phenolic compound or an aromatic amine compound in the wood.