Laccases (Lac, EC 1.10.3.2, benzendiol: oxygen oxydoreductase) belong to blue copper protein or oxidase family. Lac has been found in fungi, bacteria, and plants. The major producers of Lac are of fungi kingdom, whose diversity can be found in soil, phytopathogenic, and freshwater inhabiting ascomycetes and basidiomycetes

[104] . Lac is generally larger than peroxidases as it has a molecular weight of approx­imately 60 kDa and pi 3-6 [49]. Optimum pH for better Lac activity is found to be 3-5

[105] . Lac catalyzes four single-electron oxidations of aromatic amines and phenolic compounds such as phenolic substructure of lignin, which coincide with the reduc­tion of O2 to H2O [ 32, 98]. Indeed, it can also oxidize nonphenolic compounds under certain conditions, for example, 2,2/-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) [106], 1-hydrobenzotriazole (1-HBT) [107], and violuric acid [108]; natu­ral mediators such as 4-hydroxybenzoic acid, 4-hydroxybenzyl alcohol [109], and 3-hydroxyanthranilate [110]. Therefore, the natural mediator should be produced by organisms for the complete oxidation of lignin.

Lac is produced by almost all the white-rot fungi. Generally, it has several Lac encoding genes and secrete as multiple isoforms [49,106]. Lac contains four copper atoms of three distinct types per enzyme, and each type has a different role in the oxidation of substrate [98]. Type I copper directly involves in the reaction with the substrate. The type I copper gives a maximum absorbance at a wavelength of 610 nm, which gives to the enzyme a typical blue color. The type II copper and the two type III copper cluster are found in triangular forms. Copper II and III complexes involve in the binding, the reduction of O2 and the storage of electrons originating from the reducing substrates. The type II copper does not have absorbance in a visible range, while the type III copper has a maximum absorption at 330 nm, hence copper II and III complexes do not have any color [98]. The entire crystalline structure of Lac containing four copper atoms in the active site has been studied from T. versicolor and C. maxima [111,112]. Bourbonnaisetal. [106] reported that the white-rot fungus T. versicolor produces two laccase isozymes (I and II).

For effective biological pretreatment of lignocellulose, various white-rot fungi can be used in addition to copper ions in order to induce the secretion of Lac enzymes. In some special cases, Lac can also be induced by addition of aromatic compounds like VA and 2-5 xylidine [32]. Although Lac generally oxidizes phenolic residues of lignin, it also oxidizes non-phenolic compounds of lignin with addition of ABTS as discussed earlier. Therefore, Lac action can be induced further by addition of some special catalyst in the biological pretreatment. For some fungi such as C. subvermis — pora and Ganoderma lucidum the Lac production could be increased in the presence of lignocellulosic materials. Recently, some bacterial Lacs have also been char­acterized from Azospirillum lipoferum, Bacillus subtilis, Streptomyces lavendulae, Streptomyces cyaneus, and Marinomonas mediterranea [113].