Lignin is a complex macromolecule; its cross-linked structure renders it the most recalcitrant substance for biochemical conversion to biofuels (Vanholme et al. 2008; Ralph 2010; Carpita 2012; Vanholme et al. 2012). As with polysaccharides, lignin cleavage requires synergistic action of diverse ligninolytic enzymes, including high redox potential ligninolytic peroxidases, laccases and oxidases (Leonowicz et al. 2001; ten Have et al.

2001) .

Peroxidases, which cleave C-C and C-O bonds, are classified into heme-dependent lignin peroxidases (LiPs), manganese peroxidases (MnPs), and versatile peroxidases (VPs). Phanerochaete chrysosporium and Trametes versicolor represent efficient lignin-degrading white rot fungi; both produce LiPs and MnPs (Gold et al. 1993; Johansson et al. 1996). LiPs can directly oxidize a variety of phenolic and nonphenolic aromatic compounds via long-range electron transfer. MnPs cleave phenolic substrates depending on oxidation of Mn2+ to Mn3+ by H2O2 (Harvey et al. 1992; Wariishi et al. 1992). A LiP-like MnP enzyme, called a variable peroxidase is found in Pleurotus enryngii and Bjerkandera spp. and possesses a hybrid molecular architecture that combines different oxidation sites connected to a heme cofactor (Moreira et al. 2005; Ruiz-Duenas et al. 2007). Evolutionary analysis of peroxidases in 31 fungal genomes revealed lignin-degrading peroxidases are possessed by diverse white rot, brown rot, and mycorrhizal species in the Agaricomycetes (Floudas et al. 2012). Interestingly, molecular clock analyses places the timing of the evolution of lignin degrading peroxidases at the end of Paleozoic Era, which coincides with the end of accumulation of the vast terrestrial carbon deposits that created coal.

Laccases, which also cleave C-C and C-O bonds, are widespread, four-copper containing metalloenzymes, able to catalyze the oxidation of a variety of phenolic and lower-redox potential compounds in the presence of redox mediators (Leonowicz et al. 2001). Wood-rot fungi are the main producers of laccases, especially fungi in the class of Basidiomycetes, even though some bacteria and plants also excrete these enzymes (Leonowicz et al. 2001; Sirim et al. 2011). The laccase from Pycnoporus cinnabarinus is essential for that fungi’s lignin depolymerization ability (Eggert et al. 1997). Two laccase isozymes of T. versicolor were able to depolymerize hardwood pulp lignin in the presence of 2, 2′-azinobis (Bourbonnais et al. 1995).

Apart from peroxidases and laccases, several oxidases involved in H2O2 production and aldehyde-alcohol transformation, are indispensable in lignin decomposition (Leonowicz et al. 2001). A glucose 1-oxidase mutant of Phanerochaete chrysosporium exhibited little or no ability to degrade lignin (Ramasamy et al. 1985). Aryl alcohol oxidases (AAOs) and aryl alcohol dehydrogenase (AAD) are responsible for aromatic aldehyde-alcohol transformation during ligninolysis (Leonowicz et al. 2001). Bacterial enzymes, such as ring-fission enzymes, demethylases and p-etherases, specifically degrade the oligolignols with low molecular mass that are liberated by diverse peroxidases and/or laccase (Masai et al. 1999).

Besides fungi, a number of bacteria are able to breakdown lignin (Bugg et al. 2011). Streptomyces viridosporus T7A secretes a lignin peroxidase to depolymerize lignin. Pseudomonas putida mt-2 and Rhodococcus jostii RHA, possess comparable lignin-degrading activities. Soil bacteria such as Nocardia and Rhodococcus also mineralize lignin.

Among these lignin-degrading enzymes, laccases have been developed for larger-scale applications, such as removal of polyphenols in wine and beverages, conversion of toxic compounds and textile dyes in wastewaters, and bleaching and removal of lignin from wood and non-wood fibres (Rodriguez Couto et al. 2006). Laccases have also been used to reduce phenolic inhibitors that form during biomass pretreatment and inhibit biological fermenters (Alvira et al. 2012). Organisms that produce ligninolytic enzymes can effectively function as pretreatment agents, as much as doubling subsequent saccharification yields (Akin et al. 1995). However, these organisms tend to grow relatively slowly, with treatments taking place over days or even weeks. It appears that ligninolytic enzymes remain a relatively poorly tapped resource for facilitating biofuel production.