## 4 Discussion
The latter are considered as inducers for fungal enzymes, including lacase [30]. Similarly, the effect of sodium azide, cysteine, and EDTA have also been studied. Gallic acid, EDTA, and sodium azide (1 mM each) were found to increase lacase production in CDBT-F-G1 isolate (Figure 5h,g). However, [PERSON] and [PERSON] [31] have shown that azide, thioglycolic acid, and diethyldithiocarbamic acid inhibit secretion of lacase, whereas EDTA affected lacase secretion to a lesser extent. It is believed that small anions such as halides (excluding iodide), azide, cyanide, and hydroxide are known to bind to type-2 and type-3 copper, resulting in an interruption of internal electron transfer and thus inhibit lacase enzymes [32]. The inducing effect seen in our study may be due to increase in fungal biomass in the culture as azide ion mainly inhibits bacterial contamination in the culture. It has also beenobserved that promoter region encoding for lacases contains various recognition sites specific to xenobiotics. It is believed that aromatics, such as gallic acid, interact with the recognition sites and promote induction of lacase enzymes [33]. Further enhancement in lacase activity is possible by choosing suitable substrates. The _Pestalotiopsis_ CDBT-F-G1 isolate reported herein shows two-fold higher levels of lacase secretion as compared to other _Pestalotiopsis isolates_ (33 \(\pm\) 2 U/mL) [34]. The specific activity of lacase was found to be 26,200 U/mg with 69% recovery of enzyme which is better than the lacase recovered by 60% and 80% ammonium sulfate (65% and 63%) as reported by [PERSON] et al. and [PERSON] et al. [35; 36]. Further, acetone precipitated lacase resulted in better activity than ammonium sulfate precipitate (Table 2). In the latter study, it was reported that a high percentage of lacase retained in the protein precipitates of organic solvents, such as acetonitrile and acetone [37]. Although acetone precipitation provides better enrichment of lacase in the partially purified sample, economically, ammonium sulfate precipitation is more viable given the cost associated with precipitating agents. Accordingly, an ammonium sulfate fraction was used for further characterization of the lacase enzyme. Lacase isolated, in this study, has an optimal pH and temperature of pH 6 and 60 \({}^{\circ}\)C (Figure 6). The enzyme has a molecular mass of 43 kDa (Figure 7). Similar enzymes have been reported previously from other _Pestalotiopsis_ fungal species as well as _Ganoderma lucidum_ CDBT 1 strain with 43 kDa [18]. This enzyme isolated by precipitation techniques may be enough for the pretreatment of lignocellulosic biomass, however, additional methods of purification are necessary to obtain high-purity enzyme. It is important to note that lacase with a molar mass of 99 kDa has also been reported in _Pestalotiopsis_ species [38]. We do not see the 99 kDa increase protein in our preparations. The three possibilities as to why we did not see the second isozyme of lacase with 99 kDa in our preparations include: (i) this isozyme was not secreted; (ii) expression of this isozyme was not induced; or (iii) its concentrations were below the detectable levels. Consistent with our observation, others have also observed a single isozyme of lacase secretion by fungi cultured in the presence of phenolic compounds [39]. The other protein bands observed in partially purified preparations were co-precipitating proteins and were unlikely to be lacases because most known lacases have molecular masses larger than 30 kDa. The kinetic constants obtained for _Pestalotiopsis_ CDBT-F-G1 lacase described herein (Figure 8) was very efficient as compared to the values reported previously. The \(\mathrm{K_{m}}\) and \(\mathrm{V_{max}}\) of lacase from _Mycena purpureofusca_ were 0.3 mM and 65 uM/min, respectively. The measure of \(\mathrm{V_{max}}/\mathrm{K_{m}}\) shows the catalytic efficiency. The catalytic efficiency (\(\mathrm{V_{max}}/\mathrm{K_{m}}\)) of CDBT-F-G1 lacase was 1/min where as that of _Mycena purpureofusca_ was only 0.2/min. [40].