Occurrence and properties of the multicopper oxidases laccase and tyrosinase in lichens.

Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

The work presented in this thesis describes the occurrence and properties of two multicopper oxidases derived from lichens. Despite numerous data on laccases and tyrosinases in fungi and flowering plants, this is the first report of the occurrence of these enzymes in lichenized ascomycetes. Extracellular laccase and tyrosinase activity was measured in 50 species of lichens from different taxonomic groupings and contrasting habitats. Out of 27 species tested from suborder Peltigerineae, all displayed laccase and tyrosinase activity that correlated to each other, while activity was absent in species tested from other lichen groups. Identification of the enzymes as laccases and tyrosinases was confirmed by the ability of lichen thalli or leachates to readily metabolize substrates such as 2,2’-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS), syringaldazine and o-tolidine in case of laccase and L-dihydroxyphenylalanine (L-DOPA), Ltyrosine and epinephrine in case of tyrosinase in the absence of hydrogen peroxide. The activities of both enzymes were highly sensitive to cyanide and azide, and tyrosinase activity was sensitive to hexylresorcinol. Laccase activity had typical pH and temperature optima and an absorption spectrum with a peak at 614 nm. Tyrosinases could be activated by sodium dodecyl sulphate (SDS) and had typical tyrosinase molecular masses of approx. 60 kDa. The diversity of laccase isoforms in 20 lichen species from suborder Peltigerineae was investigated. The molecular masses of the active forms of most laccases varied between 135 and 190 kDa, although some lichens within the family Peltigeraceae had laccases with higher masses, typically varying from 200 to over 350 kDa. Most species contained one oligomeric laccase isoform. Desiccation and wounding stimulated laccase activity, while only wounding stimulated tyrosinase activity. The ability of laccases to decolorize dye is a classic attribute of laccases, and one with biotechnological potential. The ability of eight lichen species to decolourize different types of dyes was therefore tested. Interestingly, results showed that not only species belonging to suborder Peltigerineae but also species from other lichen group effectively decolourised dyes after 48 h suggesting that other oxidases appear to have ability to decolorize. Hopefully, our work could contribute to the better knowledge and application of lichen multicopper oxidases.