Effect of nutritional parameters on laccase production by the culinary and medicinal mushroom, Grifola frondosa

Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100-500 mu M Cu over the basal level (1.6 mu M Cu) and peak levels observed at 300 mu M Cu were 7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1-2 mM Cu2+. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M (r) 70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct laccase protein band (M (r) 45 kDa) was observed in cultures supplemented with 1-2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. Using 2,2'-azino-bis(ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate, the optimal temperature and pH values for laccase activity were 65 degrees C and pH 2.2, respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.