Characterization of growth conditions for production of a laccase-like phenoloxidase by Amylostereum areolatum, a fungal pathogen of pines and other conifers

Abstract

Amylostereum areolatum is a wood-degrading fungus vectored by the European woodwasp Sirex noctilio recently introduced into North America. Together they constitute a pathosystem capable of killing healthy conifers, particularly pine. The fungus kills liquid cultured cells of loblolly pine and can infect living seedlings. The fungus can be cultured on a described defined medium. Amylostereum areolatum produces a ca. 75 kDa laccase (multicopper oxidase) and conditions have been established for maximizing laccase production over 11 days. A three-step purification yields a single laccase activity band. The laccase is likely monomeric and oxidizes ABTS, catechol, 1, 8-diaminonaphthalene, guaiacol, hydroquinone, phloroglucinol, p-phenylenediamine, and syringaldazine. Its oxidation of resveratrol indicates a possible role in detoxification. Laccase activity is maximum at pH 4.0 and 30°C. It is apparently inhibited by L-cysteine, sodium azide and sodium chloride. SDS and EDTA activate the enzyme. Sodium azide has been demonstrated as a true inhibitor.