Tien et al. [63] discovered LiP in the extracellular medium of P. chrysosporium grown under nitrogen limitation. The enzyme uses H2O2 as co-factor or mediator for activity and is capable of oxidizing and/or cleaving lignin and lignin model compounds. This was supposed to be the key reaction of lignin degradation. Very few fungi are found to produce extracellular LiP [98]. P. chrysosporium, T. versicolor, Bjerkhandera sp., and T. cervina are some fungi, which can produce LiPs [32]. Indeed, LiP was found to play only a minor role in lignin degradation by T. versicolor, at least as measured by bio-bleaching of kraft pulp [99].

LiPs are monomeric homo-protein and glycol protein belonging to oxidoreduc — tase family, which specifically act on peroxide as an acceptor (peroxidases). These enzymes have molecular weight of 40 kDa and isoelectric points (pI) ranging from

2.8 to 5.3. The absorption spectrum of the native enzyme in P chrysosporium has a very distinct maximum at 406-409 nm due to the presence of a single heme group, where Fe3+ pentacoordinates with four heme tetrapyrrole nitrogen and a histidine of LiPs (protoporphyrin IX) [32, 98]. The interaction of LiPs with its substrate follows ping-pong mechanism [100]. As shown in Fig. 1.2, LiPs are oxidized by H2O2 to two-electron oxidized intermediates (LiP I) along with iron ions as Fe4+ and freerad — ical residues on tetrapyrolle. LiP I then oxidises the donor substrate by one electron, where the donor substrate, VA (3,4-dimethoxybenzyl alcohol, VA) yields second intermediate LiPs complex (LiPs II) in which iron ion is found in same oxidation state, that is, Fe+4, but there is no free radical residue on tetrapyrolle of heme and a radical cation. LiP II then oxidises a second molecules of donor substrate (VA), confers another radical cation and native form of LiP. Here the reformation of native LiP mainly depends upon the LiP II reduction step, which is a rate limiting step in catalytic cycle. Because the reduction of LiP II is a relatively slow process and LiP II is less potent than LiP I complex. Consequently, LiP II complex is long available for reaction again with H2O2 leads to inactivation of enzyme and forms LiP III com­plex (Fig. 1.2), which is characterized as a complex between LiP and superoxide. The catalytic cycle of LiP is described in Fig. 1.2. VA radical cations act as redox mediators and are capable to reduce LiP III complex back to its native form, LiP. In this LiP catalytic cycle reaction, VA radical cations (VA’+) are usually restored back after its oxidation reaction with non-phenolic compounds of lignin.

As in this catalytic cycle reaction, VA plays an important role. Three major functions of VA have been investigated so far. Firstly, VA acts as a mediator in electron-transfer reaction. Secondly, VA is a good substrate for compound II, there­fore VA is essential for completing the catalytic cycle of LiP during the oxidation of terminal substrates. Furthermore, if the inactive LiP III complex forms, the interme­diate VA^+ will be capable of reducing LiP III complex back to its native form LiP

(Fig. 1.2). Thirdly, VA prevents the H2O2-dependent inactivation of LiPs by reducing LiP II complex back to its native form LiP. Almost all the white-rot fungi synthe­size VA via de novo glucose pathway during early stage of secondary metabolism in parallel with LiP production [98].

LiPs oxidize non-phenolic and phenolic units of lignin by removing one electron and creating free radicals, which lead to chemically decompose the polymer. LiP has been shown to oxidize fully methylated lignin, lignin model compounds as well as various polyaromatic hydrocarbons. LiPs cleave selectively Ca-C|5 bond, aryl Ca bond, aromatic ring opening and demethylation in the lignin molecule [32, 98].