PROCESS FOR PRODUCING HIGH-MOLECULAR-WEIGHT COMPOUNDS OF PHENOLIC COMPOUNDS, ETC. AND USE THEREOF

Abstract: A process for macromolecularizing phenolic compounds or aromatic amine compounds by the action of a catalyst comprising an enzyme having a polyphenol oxidizing activity in the alkali region; applications of the compounds obtained by the above process to thickeners, stabilizers, coagulants, emulsifiers, dispersants, water retainers, antioxidants, adhesives, concrete admixtures, dyes, coating materials, petroleum recovering agent, soil conditioner, a blow-applied seed bearing surface soil stabilizer, deodorants, smell eliminators, agricultural chemical spreaders, feeding stuff binders, bactericides, antimicrobial agents, viral infection inhibitors, bioadhesion preventives, biotic repellents, insecticides, poultices, ink bases or wood treating agents; and method of waste water disposal, a method of deoxygenation and a method of treating wood, concrete or soil in which use is made of the above reaction.

Description: TECHNICAL FIELD The present invention relates to a method of protecting and strengthening wood through polymerization of one or more phenolic compounds or one or more aromatic amine compounds with an enzyme having polyphenol oxidizing activity. BACKGROUND ART Hitherto, it has been known that phenolic compounds etc. can be macromolecularized by utilizing an enzyme such as laccase or polyphenol oxidase produced by Basidiomycotina or Deuteromycotina (Journal of Biotechnology, 13 , 229-241, 1990 and etc. so on). However, the laccases or polyphenol oxidases produced by fungi have their optimal reaction pH in the acidic region so that the reaction must be carried out in pH region ranging from acidic to neutral in order to catalyze or accelerate the macromolecularization reaction utilizing these enzymes and in addition, the rate of the macromolecularization reaction is not high enough. Also, the natural organic compounds with which these enzymes react are mainly polyphenolic compounds and, hence, the reaction must be carried out in the pH region ranging from acidic to neutral because the optimal reaction pH of the enzyme is in the acidic region despite the fact that the polyphenolic compounds have solubilities which decrease in the pH region from acidic to neutral, resulting in a defect that it is impossible to efficiently macromolecularize polyphenolic compounds in high concentrations. Further, although many polyphenolic compounds are accelerated their autooxidation in the alkaline pH region, enzymatic oxidative macromolecularization has been carried out in the pH region ranging from acidic to neutral, resulting in a defect that the autooxidation cannot be utilized effectively. Further, it has been known that phenolic compounds, etc. can be macromolecularized with bilirubin oxidase, too, and this reaction can be utilized in the macromolecularization of lignin and dying of cotton (WO95-01426, and JP-A-6-316874). However, in the prior art using bilirubin oxidase, the enzyme-catalytic macromolecularization of phenolic compounds, etc., is carried out in the pH region ranging from acidic to neutral and therefore the macromolecularization reaction rate is not sufficiently high or there is no description suggesting that the macromolecularization reaction of phenolic compounds is accelerated in the alkaline pH region by bilirubin oxidase. OBJECT OF THE INVENTION An object of the present invention is to provide a method of treating wood utilizing a macromolecularization reaction of phenolic compounds or aromatic amine compounds. DISCLOSURE OF THE INVENTION The present inventors have made intensive investigation in order to develop a process of efficiently macromolecularizing phenolic compounds or aromatic amine compounds. As a result, they have now found that, surprisingly, use of a suitable enzyme having a polyphenol oxidizing activity in the alkaline region, particularly in the alkaline pH region not lower than pH 8, results in the achievement of efficient macromolecularization of phenolic compounds or aromatic amine compounds, thus completing the present invention. Accordingly, the present invention provides the following: 1. A method of protecting and strengthening wood through polymerization of one or more phenolic compounds or one or more aromatic amine compounds with an enzyme having polyphenol oxidizing activity, said method comprising impregnating wood with a combination comprising an enzyme having polyphenol oxidizing activity and one or more phenolic compounds or one or more aromatic amine compounds, allowing polymerization of the one or more phenolic compounds or the one or more aromatic amine compounds in the wood by said enzyme at an alkaline pH, and recovering the protected and strengthened wood. 2. The method as in 1. above, wherein the enzyme having polyphenol oxidizing activity is one or more of the enzymes selected from the group consisting of catechol oxidase, laccase, polyphenol oxidase, ascorbic acid oxidase and bilirubin oxidase. 3. The method as in 1. above, wherein the enzyme having a polyphenol oxidizing activity is an enzyme obtained by cultivating a bacterium belonging to the genus Bacillus . 4. The method as in 3. above, wherein the enzyme having a polyphenol oxidizing activity is an enzyme obtained by cultivating Bacillus licheniformis or Bacillus natto . 5. The method as in 4. above, wherein the enzyme having a polyphenol oxidizing activity is an enzyme obtained by cultivating Bacillus licheniformis SD3003 (Accession no. FERM BP-5801). 6. The method as in 1. above, wherein the enzyme having a polyphenol oxidizing activity is an enzyme obtained by cultivating a fungus belonging to the genus Myrothecium . 7. The method as in 6. above, wherein the enzyme having polyphenol oxidizing activity is an enzyme obtained by cultivating Myrothecium verrucaria or Myrothecium roridum . 8. The method as in 7. above, wherein the enzyme having polyphenol oxidizing activity is an enzyme obtained by cultivating Myrothecium verrucaria SD 3001 (Accession No. FERM BP-5520) or Myrothecium roridum SD 3002 (Accession No. FERM BP- 5523). 9. The method as in any of 1. to 8. above, wherein the phenolic compound is lignin or lignosulfonic acid. 10. A method as in any of 1. to 9. above, wherein the wood is impregnated with a combination comprizing an enzyme having polyphenol oxidizing activity, one or more phenolic compounds or one or more aromatic amine compounds, and one or more of a quinone compound, an unsaturated fatty acid, an unsaturated alcohol, and an unsaturated alkyl compound. 11. A method as in any of 1. to 9. above, wherein the wood is impregnated with a combination comprising an enzyme having polyphenol oxidizing activity, one or more phenolic compounds or one or more aromatic amine compounds, and a physiologically active substrate selected from the group consisting of an antimicrobial compound, an antiviral compound, an insecticidal compound and a biotic repellent metal ion. DETAILED DESCRIPTION OF THE INVENTION [Polyphenol Oxidase] The enzymes used for macromolecularization reaction in the present invention are enzymes having a polyphenol oxidizing activity, which are obtained by cultivating Myrothecium verrucaria SD3001 (Accession No. FERM BP-5520), Myrothecium roridum SD3002 (Accession No. FERM BP-5523) or a bacterium belonging to the genus Bacillus . However, any other enzyme having an equivalent activity of the above-mentioned enzymes may be used as well. Examples of such an enzyme include polyphenol oxidases such as catechol oxidase, laccase, polyphenol oxidase, ascorbic acid oxidase, bilirubin oxidase, produced by microorganisms, for example fungi or bacteria, or plants. Besides these enzymes, any enzyme protein that has a polyphenol oxidizing activity at alkaline pH values may also be used in the present invention. As the enzyme to be used in the present invention, having a polyphenol oxidizing activity in the alkaline pH region, those enzymes are desirable which have optimal reaction pH for polyphenol oxidation reaction in the alkaline region of not lower than pH 7.5 in order to carry out efficient macromolecularization reaction. More specifically, it is desirable that such enzymes have optimal reaction pH in the alkaline region of not lower than pH 7. 5 when the activity thereof is measured with syringaldazine that is described later. Examples of the microorganisms which produce the enzymes usable for the purposes of the present invention include the followings. As fungi, there can be cited those strains which belong to the genera falling in Deuteromycotina , i.e., Aspergillus , Botrytis , Myrothecium , Penicillium , Pestalotia , Rhizoctonia , Tricoderma , preferably Aspergillus nidulans , Botrytis cinerea , Myrothecium roridum , Myrothecium verrucaria , Myrothecium prestonii , Myrothecium leucotrichum , Penicillium sclerotiorum , Penicillium janthinellum , Pestalotia palmarum , Rhizoctonia praticola , Tricoderma resii , and Tricoderma viride . Of these, particularly preferred are Myrothecium verrucaria SD3001 (Deposited under Accession No. FBRM P-14955 at Research Institute of Biotechnologlcal & Industrial Science, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, at 1-3, Higashi 1-chome, Tsukuba-shi, Ibaragi-ken, Japan on May 29, 1995 and transferred to international deposition under Accession No. FERM BP-5520 on April 24, 1996) or Myrothecium roridum SD3002 (Deposited under Accession No. FERM P-15255 at Research Institute of Biotechnological & Industrial Science, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, at 1-3, Higashi 1-chome, Tsukuba-shi, Ibaragi-ken, Japan on October 29, 1995 and transferred to international deposition under Accession No. FERM BP-5523 on April 24, 1996). Other preferred fungi include those strains which belong to the genera falling in Basidiomycotina , i.e., Pleurotus , Lentinus , Schizophyllum , Armillariella , Flammulina , Agaricus , Coprinus , Phanerochaete , Phlebia , Lenzites , Melanoleuca , Pholiota , Stereumu , Polyporus , Polyporellus , Microporus , Fomitopsis , Pycnoporus , Trametes , Coriolus , Daedaleopsis , Rigidoporus , Fomes , Ganoderma , Trachyderma , Hymenochaete , and Inonotus , preferably Pleurotus cornucopiae , Pleurotus osteratus , Lentinus edodes , Schizophyllum commune , Armillariella mellea , Flammulina velutipes , Agaricus bisporus , Coprinus comatus , Coprinus cinereus , Coprinus congregatus , Phanerochaete chrysosporium , Phlebia radiata , Lenzites betulina , Melanoleuca verrucipes , Pholiota nameko , Stereumu hirsutum , Polyporus squamosus , Polyporellus badius , Microporus flabelliformis , Fomitopsis pinicola , Pycnoporus coccineus , Trametes orientalis , Coriolus versicolor , Coriolus hirsutus , Daedaleopsis tricolor , Rigidoporus zonalis , Fomes fomentarius , Ganoderma lucidum , Trachyderma tsunodae , Hymenochaete rubiginosa , and Inonotus mikadoi . Other preferred fungi include those strains which belong to the genera falling in Ascomycotina , i.e., Podospora , Neurospora , and Monocillium , preferably Podospora anserina , Neurospora crassa , and Monocillium indicum . Preferred bacteria include those strains belonging to Bacillus alcalophilus , Bacillus amyloliquefaciens , Bacillus brevis , Bacillus firmus , Bacillus licheniformis , Bacillus natto , Bacillus pumilus , Bacillus sphaericus , and Bacillus subtilis , preferably Bacillus licheniformis , and Bacillus . Of these, particularly preferred is Bacillus licheniformis SD3003 (Deposited under Accession No. FERM P-15383 at Research Institute of Biotechnological & Industrial Science, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, at 1-3, Higashi 1-chome, Tsukuba-shi, Ibaragi-ken, Japan on December 28, 1995 and transferred to international deposition under Accession No. FERM BP-5801 on January 28, 1997). Other preferred bacteria include those strains which belong to the genera Azospirillum , preferably Azospirillum lipoferum or those strains which belong to the genera falling in Actinomycetales , for example, Streptomyces , preferably Streptomyces antibioticus , or Aerobacter , preferably Aerobacter aerogenes . Some preferred plants which contains enzymes used in the present invention include Acer pseudoplatanum , Dioscorea , Abelmoschus , Psidium , Helianthus , potato, apple, pumpkin cucumber, wheat, alfalfa, etc. [Preparation of Enzymes] The enzymes used in the present invention can be obtained by cultivating strains belonging to the above-described microorganisms, for example fungi or bacteria, and variants thereof. Besides, they can also be prepared by utilizing genetically engineered microorganisms. That is, the enzymes can be produced by cultivating, under conditions enabling expression of enzyme proteins, host cells transformed with an expression vector which includes a DNA vector having a replication initiator codon for replicating a vector in the host organism, the vector having inserted therein a DNA sequence encoding the above-described enzyme protein together with suitable promoter, operator, and terminator DNA sequences having an enzyme expressing function in the host organism, or host cells transformed by incorporating in the host cell DNA, a DNA sequence encoding the above-described enzyme together with suitable promoter, operator, and terminator DNA sequences which have an enzyme expressing function in the host organism, followed by recovering the enzyme protein from the culture medium. The DNA fragments encoding the enzyme protein according to the present invention can be obtained by conventional methods such as the method in which cDNA or genome library from a strain belonging to the above-described microorganisms, for example fungi or bacteria, is used as an isolation source, and a target DNA fragment is identified using as a probe which is an oligonucleotide synthesized based on the amino acid sequence of the enzyme protein according to the present invention, the method in which a clone expressing the activity of an oxidase is screened, or the method in which a clone is screened which produces a protein that reacts with an antibody against the above-described enzyme protein. It is possible to prepare the enzyme protein according to the present invention by extraction from seeds, fruits, leaves or the like of the above-described plants. Further, in the cultivation of the strains belonging to fungi or bacteria and variants thereof for obtaining the enzyme protein according to the present invention, there can be used synthetic medium and nutrition medium containing organic carbon sources and organic nitrogen sources which are employed conventionally. In the case of cultivation, it is desirable that Cu 2+ ions be added in amounts of 0.001 mM to 10 mM, preferably 0.01 mM to 1 mM, as metal salt. When it is secreted outside the cells of fungi or bacteria, the polyphenol oxidase according to the present invention can be recovered from the culture medium by a well-known method. The recovery procedure includes a series of operations such as removal of cells from the culture medium by centrifugation, filtration or membrane separation, and chromatography, for example ion exchange chromatography. Also, membrane concentration with ultrafiltration membrane is effectively employed. When it is accumulated inside the cells of fungi or bacteria or when it exists inside plant tissues, the enzyme protein can be recovered from the microbial cells or plant tissues by a well-known method. The recovery procedure includes a series of operations such as mechanical rupture of the tissue by homogenization, isolation and extraction of an enzyme protein solution by centrifugation, filtration or membrane separation, and chromatography, for example ion exchange chromatography. Also, membrane concentration with ultrafiltration membrane is effectively employed. [Measurement method of Activity] In the present invention, the measurement of polyphenol oxidizing activity of the enzyme protein having a polyphenol oxidizing activity was carried out by conducting the reaction in an aqueous solution containing 20 ppm of syringaldazine and 100 mM Tris-HCl buffer or potassium phosphate buffer at an optimal reaction pH at 20°C and measuring the absorbance at 525 nm. The amount of activity in which 1 pmol/minute of syringaldazine is oxidized was defined as 1 unit (hereafter, abbreviated as "U"). [Macromolecularization Method and Its Use] In the process of producing phenolic compounds or aromatic ermine compounds having increased molecular weights used according to the present invention, the concentration of phenolic compounds or aromatic amine compounds is 0.01 to 90%, preferably 1 to 80%. The reaction temperature is 0 to 150°C, preferably 0 to 100°C. Further, the reaction pH is 7.0 to 12, preferably 7.5 to 10. The activity concentration of the enzyme to be used is 1 to 10,000 U/liter, preferably 10 to 2,000 U/liter. It is desirable that the enzyme activity concentration be adjusted depending on the purpose. That is, when more speedy macromolecularization and gelation or solidification is contemplated to be achieved, the reaction will be carried out at higher activity concentrations. On the other hand, when the reaction is carried out at lower activity concentrations, milder macromolecularization reaction will proceed, giving rise to a more homogeneous macromolecule solution as a liquid substance, and when the reaction is continued further, mild gelation reaction will proceed throughout the reaction mixture. When a suitable degree of macromolecularization is reached, the reaction can be terminated by the addition of alkali or alkali salts, such as NaOH, NH 3 , Na 2 CO 3 , and CaCO 3 , addition of acids such as hydrochloric acid, sulfuric acid, and nitric acid, addition of known enzyme inhibitors, or heat treatment such as that at 100°C for 15 minutes. The gelled phenolic compounds or aromatic amine compounds can optionally be molten again by heating at 50 to 230°C. Also, it is possible to obtain phenolic compounds or aromatic amine compounds having very high molecular weights as a solution by addition of hot water or the like after the heat melting to disperse or dissolve the compounds above. In order to accelerate the thermal curing, it is also possible to add polyols such as furfuryl alcohol, sugars and etc. In order to have a physiologically active substance contained in the macromolecular compound to obtain an immobilized physiologically active substance or substance with a controlled release of physiologically active substance, it is also possible to carry out the macromolecularization with an antimicrobial compound, antiviral compound, a biotic repellent compound, an insecticidal compound or a metal ion coexisting, or to add an antimicrobial compound, antiviral compound, a biotic repellent compound, an insecticidal compound or a metal ion after the macromolecularization. As the antimicrobial compound, antiviral compound, biotic repellent compound, insecticidal compound or metal ion used for this purpose, there may be used many substances that have been known heretofore. The macromolecularization reaction used according to the present invention uses as an oxidation catalyst an enzyme having a polyphenol oxidizing activity and the oxygen in the air as an oxidizer. Further, when production of a large amount of macromolecule is contemplated, operations such as mechanical stirring of the reaction mixture and addition of air or oxygen to the reaction system are effective. Also, it is possible to carry out the reaction of the present invention in which oxygen is used as an oxidizer and the reaction in which hydrogen peroxide is used as an oxidizer simultaneously by adding to the reaction mixture peroxidase and hydrogen peroxide, or instead of hydrogen peroxide, an oxidase which can generate hydrogen peroxide and a substrate thereto. [Phenolic Compounds or Aromatic Amine Compounds] As the phenolic compounds or aromatic amine compounds to be macromolecularized in the present invention, there may be used any compound as far as the enzyme used in the present invention can oxidize it. Specific examples of such phenolic compounds or aromatic amine compounds include lignin, lignosulfonic 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, vanillic acid, vanillyl alcohol, ascorbic acid, 1,2-dihydroxynaphthalene, 2,3-dihydroxpnaphthalene, 6,7-dihydroxy-2-naphthalenesulfonic acid, anthrarobin, alizarin, quinizarin, o-phenylenediamine, p-phenylenediamine, 3,4-diaminobenzophenone, o-anisidine, p-anisidine, o-aminophenol, p-aminophenol, 1,2-diamino-anthraquinone, and 1,4-diaminoanthraquinone. Compounds other than these may also be used as a raw material for macromolecules or as a catalyst for the macromolecularization reaction as far as such compounds are substances that the enzyme used in the present invention can oxidize. Examples of such compounds include ABTS (2,2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid)), bilirubin, isoascorbic acid, quercetin, rutin, guaiacol, 4-methoxyphenol, biphenol, 4,4'-ethylene-dianiline, 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-methoxybenzoate, 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-cyclobuten-1,2-dione, 3-(3,4-dihydroxyphenyl)-L-alanine, 2-amino-3-hydroxypyridine, 3-amino-2-methoxydibenzofurane, 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-diemethoxybenzoic acid, veratric acid, 3,4-dimethoxybenzyl alcohol, 3,4-dimethoxyphenothylamine, (3,4-dimethoxy-phenyl)acetic acid, (3,4-dimethoxyphenyl)acetonitrile, 4-allyl-2-methoxyphenol, 2-methoxy-4-propenylphenol, 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, purpurogallin, 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. Also, in order to control the physical properties of the macromolecules, it is possible to use a plurality of such phenolic compounds or aromatic amihe compounds in combination. Upon producing macromolecularized phenolic compounds or aromatic amine compounds according to the present invention, quinone compounds may coexist which can be macromolecularized in a similar reaction path. Examples of such quinone compounds include anthraquinone-2-sulfonic acid, anthraquinone-1,5-disulfonic acid, anthraquinone-2,6-disulfonic acid, anthraquinone-2-carboxylic acid, 1-aminoanthraquinone, 2-aminoanthraquinone, anthrarufine, aminonaphthoquinone, 1,8-dihydroxyanthraquinone, camphorquinone, dehydroascorbic acid, 2-hydroxy-1,4-naphthoquinone, isatin, 5-nitroisatin, and various anthraquinone dyes. Also, it is possible to carry out air oxidation and macromolecularization simultaneously with enzymatic reaction under the coexistence of autooxidated substances, e.g., unsaturated fatty acids such as oleic acid and rinolic acid or unsaturated alcohols such as oleyl alcohol, or unsaturated alkyls such as squalene. Of the macromolecularized phenolic compounds or aromatic amine compounds produced according to the present invention, particularly macromolecules of natural substances or, derivatives thereof, such as lignin, lignosulfonic acid, humic acid, nitrohumic acid, tannin, catechin, gallic acid, urushiol, hesperidin, and hinokitiol, are highly useful because they are highly safe to environment as well as to humans so that making the best of their characteristics as high-molecular-weight compounds, they can be utilized in the field of wood treating agents. It should be noted that in this application, various additive components usually used in each field can be employed in combination. Further, in this field of application, it is possible to develop applications of higher functions of the high-molecular-weight compounds by macromolecularizing natural or non-natural phenolic compounds or aromatic amine compounds under milder reaction conditions in the production process of the present invention to control the physical properties such as viscosity, adhesion, water retention, water solubility, water resistance, resilience, and strength or physiological effects. In accordance with the present invention, impregnation of the enzyme having a polyphenol oxidizing activity together with phenolic compounds or aromatic amine compounds into wood and macromolecularization, in the wood, of the phenolic compounds or aromatic amine compounds and in addition the polyphenol compounds such as lignin already contained in the wood enables improvement in workability in a drying step after the wood impregnation treatment, improvement in the strength of wood which was decreased due to lignin decomposition by wood boiling treatment or high temperature steam injection treatment, and improvement in the effect of preventing wood cracking upon drying or freezing, prevention of growth of microorganisms due to maintenance or improvement in anaerobic environment in wood. Also, in accordance with the present invention, by allowing polyphenol oxidase and a dye or its precursor on which the polyphenol oxidase can act, to act on wood, it is possible to produce a coloring substance in the wood or to co-macromolecularize the coloring substance and polyphenol compounds such as lignin already contained in the wood so that firmer dying or coloring treatment of wood can be achieved. In the above-described wood dying or coloring treatment, many polyphenol oxidases are known to bleach lignin, which is a coloring substance contained in wood and, hence, the wood dying or coloring treatment according to the present invention is very useful since it allows enzymatic bleaching and dying or coloring treatment simultaneously, thus reducing the number of process steps and improving the color tone. BEST MODE FOR CARRYING OUT THE INVENTION Hereafter, the present invention will be described more concretely by representative examples which, however, are merely exemplary and the present invention should not be construed as being limited thereto. In the following examples, the molecular weight analysis for macromolecularized phenolic compounds or aromatic amine compounds was carried out by HPLC using 50 mM of potassium phosphate buffer (pH 7.0) or 0.1 mM of sodium sulfate aqueous solution as an eluant, Shodex RI (differential refractive index detector, manufactured by Showa Denko) as a detector, and Shodex PROTEIN KW-802.5 (tandem, manufactured by Showa Denko) or a combination of Shodex PROTEIN KW802.5 (manufactured by Showa Denko) with Shodex OHpak SB-804HQ (manufactured by Showa Denko) as a column. Example 1: Cultivation and partial purification, concentration In a 500 ml flask as a cultivation apparatus charged with 100 ml of a culture medium containing 0,134% Na 2 HPO 4 ·12H 2 O, 0.03% KH 2 PO 4 , 1% maltose, 1% peptone, 0.1% yeast extracts, 0.05% MgSO 4 ·7H 2 O, 0.1 mM CuSO 4 , 1 mM MnCl 2 , and 2 mM CaCl 2 , adjusted to pH 7.8 by addition of 20% Na 2 CO 3 , was inoculated with Bacillus licheniformis SD3003 (Accession No. FERM BP-5801). After cultivation with shaking at 50°C for 16 hours, the cultivation temperature was lowered to 35°C and the cultivation was continued for 3 days. After the cultivation, the culture broth was centrifuged at 4°C to obtain a culture broth eliminated the bacillus. To further purify and concentrate this, ammonium sulfate fractionation was effective and at 20 to 60 % saturation ammonium sulfate concentration, most polyphenol oxidase activity was recovered as precipitation. The obtained ammonium sulfate precipitation was dialyzed against 10 mM Bis-tris/HCl buffer solution (pH 7.0). For further purification and concentration, there was used ultrafiltration membrane to obtain a partially purified concentrated aqueous solution (0.8 U/ml) in the fractions corresponding to molecular weights of 10,000 to 100,000. Example 2: Cultivation and concentration In a culture cistern containing a 3 liter culture medium consisting of 0.5% glucose, 0.1% NaNO 3 , 1.34% Na 2 HPO 4 ·12H 2 O, 0.3% KH 2 PO 4 , 0.1% NaCl, 0.2% peptone, 20 ppm yeast extracts, 0.01% MgSO 4 ·7H 2 O, and 0.1 mM CuSO 4 , adjusted to pH 8 by addition of 10% NaOH, was inoculated with Myrothecium verrucaria SD3001 (Accession No. FERM BP-5520) and cultivation with shaking was carried out at 28°C for 3 days. After the cultivation, centrifugation at 4°C was carried out to obtain 2.5 liters of culture broth eliminated the bacillus. . Then, a part of the culture broth was concentrated to a fraction having a molecular weight of not smaller than 10,000 by using Minitan ultrafiltration system (manufactured by Millipore Co.) with Minitan filter packet (CAT. No.: P.TGCOMP04 manufactured by Millipore Co.). Furthermore, after further dialysis against 200 ppm NH 4 HCO 3 , this was lyophilized to obtain a crude product as a freeze-dried product. The freeze-dried product had a polyphenol oxidase activity of 15 U/mg. An aqueous solution of the freeze-dried product showed an absorption maximum near 600 nm, which is specific to copper-containing proteins. Example 3: Cultivation and concentration In a culture cistern containing a 3 liter culture medium consisting of 0.5% glucose, 0.1% NaNO 3 , 1.34% Na 2 HPO 4 ·12H 2 O, 0.3% KH 2 PO 4 , 0.1% NaCl, 0.2% peptone, 20 ppm yeast extracts, 0.01% MgSO 4 ·7H 2 O, and 0.1 mM CuSO 4 , adjusted to pH 8 by addition of 10% NaOH, was inoculated with Myrothecium roridum SD3002 (Accession No. FERM BP-5523) and cultivation with shaking was carried out at 28°C for 3 days. After the cultivation, centrifugation at 4°C was carried out to obtain 2.5 liters of culture broth eliminated the bacillus. Then, a part of the cultures broth was concentrated to a fraction having a molecular weight of not smaller than 10,000 by using Minitan ultrafiltration system (manufactured by Millipore Co.) with Minltan filter packet (CAT. No.: PTGCOMPO4 manufactured by Millipore Co.). Furthermore, after further dialysis against 200 ppm NH 4 HCO 3 , this was lyophilized to obtain a crude product as a freeze-dried product. The freeze-dried product had a polyphenol oxidase activity of 10 U/mg. An aqueous solution of the freeze-dried product showed an absorption maximum near 600 nm, which is specific to copper-containing proteins. Example 4: Macromolecularization reaction Macromolecularization reactions [4-1] to [4-3] described below were carried out using the partially purified concentrated aqueous solution described in Example 1, and macromolecularization reaction [4-4] was performed using commercially available polyphenol oxidase (obtained from TaKaRa). [4-1]: 1 ml of reaction mixture was prepared which contained 20% (W/V) sodium lignosulfonate (obtained from Aldrich Chemical Company, Inc.) and the partially purified concentrated aqueous solution as a polyphenol oxidase in an activity concentration of 300 U/liter, and was shaken at a reaction temperature of 70°C and at 100 rpm in a glass test tube to allow reaction. The pH of the reaction mixture was adjusted to pH 7.5 with a minute amount of sulfuric acid. After the initiation of reaction, the color tone of the reaction mixture turned denser immediately and after 6 hours, there was observed significant progress of macromolecularization and after 20 hours, most of the reaction mixture was solidified. [4-2]: Also, in the case where the reaction was carried out with changing the activity concentration of polyphenol oxidase to 60 U/liter, the whole reaction mixture was highly viscous liquid after 20 hours from the initiation of reaction, and the molecular weight of the product increased according as the viscosity increased. After continuing additional a 20 hour reaction, there was observed partial solidification of the reaction mixture. Samples for molecular weight analysis were prepared by taking out a part of the reaction mixture and heating in a water bath at about 100°C for 15 minutes to terminate the reaction. [4-3]: Also, when lignin (alkali) (obtained from nacalai tesque ) was used in a concentration of 20% (W/V) instead of lignosulfonic acid and the reaction was carried out at an activity concentration of polyphenol oxidase of 300 U/liter at pH 7.5, the whole reaction mixture became highly viscous liquid after 24 hours from the initiation of reaction. [4-4]: 100 ml of reaction mixture was prepared, which contained 20% (W/V) sodium lignosulfonate and a commercially available polyphenol oxidase as a polyphenol oxidase in an activity concentration of 300 U/liter and was shaken at a reaction temperature of 25°C and at 100 rpm in a 500 ml flask to allow reaction. Since the commercially available polyphenol oxidase used herein was shown to be acid enzyme having an optimal reaction pH of 6 to 7 as measured using syringaldazine, the pH of the reaction mixture in the macromoiecularization reaction was adjusted to pH 6.5 with a minute amount of sulfuric acid. After the initiation of reaction, the color tone of the reaction mixture turned denser immediately, but it took about 80 hours of reaction before most of the reaction mixture could be solidified. Example 5: Macromolecularization reaction Using the freeze-dried products described in Example 2, there were carried out macromolecularization reactions [5-1] to [5-3] below. [5-1]: 100 ml of reaction mixture was prepared, which contained 20% (W/V) sodium lignosulfonate and the freeze-dried product as a polyphenol oxidase in a concentration of 20 ppm (300 U/liter) and was shaken at a reaction temperature of 25°C and at 100 rpm in a 500 ml flask to allow reaction. The pH of the reaction mixture was adjusted to pH 9 with NaOH. After the initiation of reaction, the color tone of the reaction mixture turned denser immediately and after 3 hours, there was observed significant progress of macromolecularization, and after 10 hours, most of the reaction mixture was solidified. Also, in the case where the reaction was carried out by changing the amount of polyphenol oxidase added to 4 ppm, after 20 hours the whole reaction mixture became highly viscous liquid, and after continuing additional a 20 hour reaction, there was observed partial solidification of the reaction mixture. Samples for molecular weight analysis were prepared by taking out a part of the reaction mixture and heating in a water bath at 90°C for 5 minutes to terminate the reaction. [5-2]: Also, in the case where lignin (alkali) was used in a concentration of 20% (W/V) instead of lignosulfonic acid, the reaction was carried out at an activity concentration of polyphenol oxidase of 300 U/liter at pH 9 and at a reaction temperature of 25°C. After the initiation of the reaction, the color tone of the reaction mixture turned denser immediately, the macromolecularization reaction proceeded, and after 24 hours, most of the reaction mixture was solidified. [5-3]: Using 20% (W/V) sodium lignosulfonate, the reaction was carried out at a reaction temperature of 25°C in an activity concentration of polyphenol oxidase of 300 U/liter, with the pH of the reaction mixture adjusted to 7 with a small amount of sulfuric acid. After the initiation of the reaction, the color tone of the reaction mixture turned denser immediately. However, there was observed no solidification of the reaction mixture. Example 6: Macromolecularization reaction Using the freeze-dried products described in Example 3, there were carried out macromolecularization reactions [6-1] to [6-2] below. [6-1]: 100 ml of reaction mixture was prepared, which contained 20% (W/V) sodium lignosulfonate and the freeze-dried product as a polyphenol oxidase in a concentration of 30 ppm (300 U/liter) and was shaken at a reaction temperature of 25°C and at 100 rpm in a 500 ml flask to allow reaction. The pH of the reaction mixture was adjusted to pH 9 with NaOH. After the initiation of reaction, the color tone of the reaction mixture turned denser immediately and after 3 hours, there was observed significant progress of macromolecularization, and after 10 hours, most of the reaction mixture was solidified. [6-2]: Also, in the case where lignin (alkali) was used in a concentration of 20% (W/V) instead of lignosulfonic acid, the reaction was carried at an activity concentration of polyphenol oxidase of 300 U/liter at pH 9 and at a reaction temperature of 25°C. After the initiation of the reaction, the color tone of the reaction mixture turned denser immediately, and after 24 hours, most of the reaction mixture was solidified. Comparative Example: Macromolecularization reaction using a known enzyme 100 ml of reaction mixture was prepared, which contained 20% (W/V) lignin (alkali) and a commercially available bilirubin oxidase (freeze-dried product) (obtained from Sigma) as a polyphenol oxidase in a concentration of 300 U/liter and the reaction mixture was shaken at a reaction temperature of 25°C and at 100 rpm in a 500 ml flask to allow reaction. Since the bilirubin oxidase used herein was shown to have an optimal reaction pH of 8 to 9 as measured using syringaldazine, the macromolecularization reaction was carried out at pH 8.5 which was the pH of the reaction mixture without adjustment. After the initiation of reaction, there was observed progress of macromolecularization. However, it needed about 50 hours from the initiation of reaction to solidify most of the reaction mixture. Example 7: Antifungal properties (illustrative) Solid (gel-like) lignosulfonic acid obtained by a reaction similar to the macromolecularization reaction described in [5-1] in Example 5 was sliced to small pieces (10 mm × 10 mm × 3 mm), which were placed in the center of an L agar plate. Then, spores of Aspergillus orizae AHU7134 were spread over the whole surface of a culture medium of a plate and cultivated at 28°C for 4 days. As a result, there was observed inhibition of growth of the fungus in the range of the upper part of and about 2 mm from the periphery of each small piece of lignosulfonic acid, which indicated that it was useful as an antifungal agent. Example 8: Antifungal properties (illustrative) To a 20% (W/V) aqueous solution of sodium lignosulfonate was added 200 ppm of hinokitiol (obtained from Tokyo Kasei Kogyo Co., Ltd.) and the resulting mixture was heated at 90°C to melt hinokitiol, followed by suspending hinokitiol using a vortex mixer and cooling the suspension to 25°C. To the thus-obtained raw material for macromolecularization reaction was added the freeze-dried product described in Example 2 as a polyphenol oxidase in an activity concentration of 300 U/liter, and the mixture was subjected to macromolecularization reaction at a reaction temperature of 25°C and at pH 9 to obtain solidified (gel-like) lignosulfonic acid containing hinokitiol. The solid product was sliced to small pieces (10 mm x 10 mm x 3 mm), which were placed in the center of an L agar plate. Then, spores of Aspergillus orizae AHU7134 were spread over the whole surface of a culture medium of a plate and cultivated at 28°C for 4 days. As a result, there was observed inhibition of growth of the fungus in the range of the upper part of and about 10 mm from the periphery of each piece of lignosulfonic acid, which indicated that it was useful as an antifungal agent and as an antifungal agent retainer. Also, to a 20% (W/V) aqueous solution of sodium lignosulfonate was added (+)-catechin·H 2 O (obtained from Sigma) in a concentration of 5,000 ppm and the freeze-dried product described in Example 2 as a polyphenol oxidase in an activity concentration of 300 U/liter. Then, the mixture was subjected to macromolecularization reaction at a reaction temperature of 25°C and at pH 9 to obtain solidified (gel-like) lignosulfonic acid containing catechin. The antifungal properties of the solid product was tested in the same manner as in the above-described example and as a result, there was observed inhibition of growth of the fungus in the range of the upper part of and about 8 mm from the periphery of each piece of lignosulfonic acid, which indicated that it was useful as an antifungal agent and as an antifungal agent retainer. Example 13: Wood treatment A reaction mixture was prepared which contained the freeze-dried product described in Example 2 as a polyphenol oxidase in a concentration of 4 ppm and the reaction mixture was allowed to react in the same manner as the macromolecularization reaction described in [5-1] in Example 5 to obtain a water-soluble macromolecularized lignosulfonic acid (20% (W/V)). To note, an operation, such as heating, for terminating the macromolecularization reaction was not carried out. An aqueous solution of the macromolecularized lignosulfonic acid was diluted to 10 fold. Using the resulting solution as a wood treating agent, injection treatment with the agent was conducted on a cryptomeria log (unseasoned wood of 3 cm in diameter and 20 cm in length) which was treated in aqueous 0.1% Tween 80 solution at 60°C for 16 hours after removal of its bark. The injection treatment was carried out by the Bethell process involving pressure reduction to 720 mmHg and pressurization to 10 kg/cm 2 . The log samples subjected to injection treatment with non-macromolecularized lignosulfonic acid and macromolecularized lignosulfonic acid, respectively, (two in each case) were set on the soil about 20 cm from the circumference of the nest of termite and left to stand for 2 months. Then, observation was made of termite preventing effect by the treatment with the agent. As a result, slight eating by termite occurred in the log samples treated with the non-macromolecularized lignosulfonic acid whereas no eating by termite was observed in the log samples treated with the macromolecularized lignosulfonic acid, the latter samples maintaining (homogeneity smoothness, luster) of log surface. Example 14: Wood treatment To a preparation obtained by adjusting an aqueous 1% p-phenylenediamine dihydrochloride (manufactured by Kanto Kagaku Co., Ltd.) to pH 9 with a small amount of NaOH was added the freeze-dried product described in Example 2 as a polyphenol oxidase in a concentration of 0.2 ppm. The'preparation was immediately coated on a oak plate. Coloring reaction proceeded quickly on the surface and inside (depth: up to about 3 mm) of the plate to give rise to a plate material firmly colored in dark brown.

Claims: A method of protecting and strengthening wood through polymerization of one or more phenolic compounds or one or more aromatic amine compounds with an enzyme having polyphenol oxidizing activity, said method comprising impregnating wood with a combination comprising an enzyme having polyphenol oxidizing activity and one or more phenolic compounds or one or more aromatic amine compounds, allowing polymerization of the one or more phenolic compounds or the one or more aromatic amine compounds in the wood by said enzyme at an alkaline pH, and recovering the protected and strengthened wood. The method as claimed in claim 1 wherein the enzyme having polyphenol oxidizing activity is one or more of the enzymes selected from the group consisting of catechol oxidase, laccase, polyphenol oxidase, ascorbic acid oxidase and bilirubin oxidase. The method as claimed in claim 1, wherein the enzyme having a polyphenol oxidizing activity is an enzyme obtained by cultivating a bacterium belonging to the genus Bacillus . The method as claimed in claim 3, wherein the enzyme having a polyphenol oxidizing activity is an enzyme obtained by cultivating Bacillus licheniformis or Bacillus natto . The method as claimed in claim 4, wherein the enzyme having a polyphenol oxidizing activity is an enzyme obtained by cultivating Bacillus licheniformis SD3003 (Accession no. FERM BP-5801). The method as claimed in claim 1, wherein the enzyme having a polyphenol oxidizing activity is an enzyme obtained by cultivating a fungus belonging to the genus Myrothecium. The method as claimed in claim 6, wherein the enzyme having polyphenol oxidizing activity is an enzyme obtained by cultivating Myrothecium verrucaria or Myrothecium roridum. The method as claimed in claim 7, wherein the enzyme having polyphenol oxidizing activity is an enzyme obtained by cultivating Myrothecium verrucaria SD 3001 (Accession No. FERM BP-5520) or Myrothecium roridum SD 3002 (Accession No. FERM BP- 5523). The method as claimed in any of claims 1 to 8, wherein the phenolic compound is lignin or lignosulfonic acid. A method as claimed in any of claims 1 to 9, wherein the wood is impregnated with a combination comprizing an enzyme having polyphenol oxidizing activity, one or more phenolic compounds or one or more aromatic amine compounds, and one or more of a quinone compound, an unsaturated fatty acid, an unsaturated alcohol, and an unsaturated alkyl compound. A method as claimed in any of claims 1 to 9, wherein the wood is impregnated with a combination comprising an enzyme having polyphenol oxidizing activity, one or more phenolic compounds or one or more aromatic amine compounds, and a physiologically active substrate selected from the group consisting of an antimicrobial compound, an antiviral compound, an insecticidal compound and a biotic repellent metal ion.

Inventors: ECHIGO TAKASHI, OHNO RITSUKO, ECHIGO, TAKASHI, OHNO, RITSUKO, ECHIGO, Takashi, c/o Showa Denko K.K., OHNO, RITSUKO, C/O SHOKO CO., LTD.
Applicants: SDS BIOTECH CORP, SDS BIOTECH K.K.
Application year: 1997
Main classification: C12P7
IPC codes: C09K3, C12R1, C12P13, C09K15, A61P31, B27K3, A61K31, A23K1, A01G7, A23L3, C12P7, B09C1, C08G83, C09K17, C09K101