Abstract
Stereum purpureum was stationarily grown in PD medium at 29C for 20 days, and the laccase was purified from its culture filtrate by column chromatographies on DEAE-cellulose, DE-52, and Sephadex G-100. The purified enzyme was homogeneous in the electrophoresis as well as the ultracentrifuge, and the degree of purification was about 90-fold over the initial filtrate. The relative reaction rates for p-phenylenediamine, guaiacol, pyrogallol, catechol and hydroquinone were estimated to be 100, 86.9, 80.7, 79.7 and 45, respectively. The Km values for these substrates were 0.833, 0.760, 0.101, 2.63 and 2.0mM, respectively. The enzymatic reaction was strongly inhibited by several reducing agents, such as sodium hydrosulfite, cysteine and ascorbic acid, but no effects were observed by chelating agents, such as EDTA, αα'-dipyridyl and o-phenanthroline. In the feeding experiment using healthy nursery stocks of apple (cv. Fuji, 2 years old), the woody tissues showed browning at the extremely high concentration of laccase (5, 000 units). On the other hand, slight browning was observed on the cut-surface of the woody pieces, when the enzymatic solution (1, 000 units) was put on them. On the contrary, remarkable browning was recognized on the cut-surface of the boiled woody pieces at the lower concentration (100 units). It can be thought from these results that the laccase produced by the pathogen may participate in tissue-browning accompanied by the development of disease.
