T1 site function as electron acceptor site where the enzyme catalyses four 1 e-oxidation of a reducing substrate (Huang et al. 1999a, 1999b). The T2/T3 is the trinuclear site where molecular oxygen is reduced by accepting the electrons from T1 site. This nature of copper site in laccase has been elucidated by spectroscopy studies and DFT.
It has been reported by Bourbonnais et al. 1995, 1998 that non phenolic compounds and lignin are oxidized by laccase in presence of mediators such as 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), I hydroxybenzotriazole (HBT) and 3 hydroxyanthranilic acid
At T2 and T3 copper ion site, one oxygen atom of the dioxide anion bounds, and other oxygen atom is bound with anther copper ion of T3. After that peroxide intermediate undergoes a second 2e- process to produce a native intermediate that has three copper centers in the trinuclear site which is mutually bridged by the product of full O2 reduction with at least one Cu-Cu distance of 3.3Å.
It has been reported after studying combination of model studies and calculation that three copper centers in the trinuclear cluster all are bridged by m3 oxo ligand 19. This m3 oxo ligand has been attributed in providing a relatively stable structure of the native intermediate that serves as the thermodynamic driving force for the 4e process of O2 reduction, and also, provides efficient electron transfer (ET) pathways from the T1 site to the other copper centers in the trinuclear cluster. 19
Laccases are the enzymes which are secreted out in the medium extra cellularly by several fungi 25 Basidiomycetes and Saprotrophic fungi are the most widely known species that produce substantial amount of laccase in changeable quantity 42. Several factors influence laccase production such as type of cultivation (submerged or solid state), carbon limitation, and nitrogen source 45. In case of Pycnoporus cinnabarinus, laccase was the only ligninolytic enzyme which degrades lignin 17
Influence of carbon and nitrogen source
Nitrogen source in the medium plays important role in enhancing laccase activity. Most commonly used nitrogen sources are yeast extract, peptone, (NH4)2SO4 and NaNO3. According to some findings laccase production was found to be triggered by nitrogen depletion 46. It was also reported that low carbon to nitrogen ratio elevates the laccase activity while others show that high laccase activity achieved by using high carbon to nitrogen ratio 49