Departmental Bulletin Paper 〈Originals〉Purification and partial characterization of laccase from the soft-rot fungus Graphium sp. M-1-9a

田中, 裕美  ,  山本, 貴義  ,  本田, 克樹  ,  板倉, 修司

[Synopsis]An extracellular laccase was isolated, purified, and partially characterized from the culture medium of Grphium sp. M-1-9, which causes soft rot in Japanese beech wood. Laccase producted by this fungus in cultures supplemented with copper sulfate was purified 60-fold by ammonium-sulfate precipitation, Sephadex G-100 gel-filtration chromatography, DEAE Sepharose ion-exchange chromatography, and Superdex G-75 gel-filtration chromatograpgy, with a yield of 3.3%. Native and SDS-PAGE showed an active band with a molecular mass of approximately 67,000. Purified laccase maintained 70-80% stability at pH 6-10 for 180 min and at 30-40°C for 120 min. The optimum pH for the oxidation of 2,2´-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was pH 3, while that for the oxidation of 2,6-dimethoxyphenol (DMP), guaiacol, and syringaldazine was pH 6. Kinetic constants (Km and kcat/km) showed that 2,6-DMP and syringaldazine were more effective substrates than ABTS and guaiacol. Enzyme activity was completely inhibited by sodium azide or L-cysteine at a concerntration of 1 mM. Laccase activity was also inhibited by 5 mM SDS.[要約]ブナ材に軟腐朽を起こすGraphiumsp. M-1-9を、硫酸銅を添加したブナ木片培地で培養し、ラッカーゼを分離・精製し、その諸性質を検討した。硫安沈殿、ゲルロ過クロマトグラフィー、イオン交換クロマトグラフィーにより60倍に精製されたラッカーゼの収量は3.3%であった。SDS-PAGEにより、分子量は約67000であった。精製したラッカーゼはpH 6-10では180分間、また30-40℃では120分間、70-80%の活性を維持した。最適pHは基質により異なっており、2,2ʹ-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)ではpH 3、2,6-dimethoxyphenol(DMP)、guaiacol,、syringaldazineではpH 6であった。Kmとkcat/Kmより2,6-DMP とsyringaldazineは、ABTS とguaiacolより効率的な基質であった。酵素活性は1 mM.のアジ化ナトリウムあるいはL-システイン、5 mM SDSで阻害された。

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