Process for producing high-molecular-weight phenolic compounds with myrothecium

#This application was filed under 35 USC 371 as the national phase of PCT/JP97/01694 filed May 20, 1997.

#The present invention relates to a process for enzymatic macromolecularization of phenolic compounds or aromatic amine compounds and to applications of the macromolecules obtained by the process.

#More particularly, the present invention provides a process for producing phenolic compounds or aromatic amine compounds which have increased molecular weights by reacting phenolic compounds or aromatic amine compounds with an enzyme having a polyphenol oxidizing activity in the alkaline pH region, and applications of the reaction method of increasing the molecular weight of phenolic compounds or aromatic amine compounds utilizing the above-described catalytic activity of the enzyme in the alkali regions to obtain thickeners, stabilizers, coagulants, emulsifiers, dispersants, water retainers, antioxidants, adhesives, concrete admixtures, dyes, deodorants, smell eliminators, coating materials, petroleum recovering agents, soil conditioners, blow-applied seed bearing surface soil stabilizers, agricultural chemical spreaders, feeding stuff binders, bactericides, antimicrobial agents. viral infection inhibitors, bioadhesion preventives, biotic repellents, insecticides, poultices, ink bases and wood treating agents; a method of producing these various agents; a method of waste water disposal; a method of deoxygenation; and methods of treating wood, concrete and soil, respectively.

#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.

#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% Na2HPO4.12H2O, 0.03% KH2PO4, 1% maltose, 1% peptone, 0.1% yeast extracts, 0.05% MgSO4.7H2O, 0.1 mM CuSO4, 1 mM MnCl2, and 2 mM CaCl2, adjusted to pH 7.8 by addition of 20% Na2CO3, 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.