In the last few years laccases have been identified as important enzymes for application in the environment sector as well as for production of high value aromatics [4, 26]. Almost all laccases are produced at low levels in the native fungi. Multiple isozymes of laccase have also been reported in numerous fungi making the study of individual enzymes difficult. Cloning and expression of genes provides an opportunity to overproduce and study these enzymes individually. Based on our previous studies [15, 18], wherein a laccase was purified from C. bulleri and investigated for its application in degradation of textile dyes, the cDNA encoding this enzyme was isolated in the present study and expressed in P. pastoris. Although many white-rot fungi exhibit multiple isozymes of laccase, we and others  have only observed one laccase in C. bulleri. To isolate the full length cDNA encoding this laccase, RLM-RACE technique was used. This method has been found to be very useful in isolating gene sequences using primers from the known and the conserved regions  of the genes. Primers were designed in the present study, based on the previously sequenced 435 bp sequence , and used to obtain the complete coding sequence through the primer walking technique. A comparison of the complete protein sequence with other laccases indicated high similarity to basidiomycete laccases, especially in the copper binding regions, with all His and Cys residues conserved. The internal peptide sequences reported  for the nLac were identified in the rLac indicating that the cloned gene was that of the enzyme studied earlier. The reported  N-terminus sequence of the laccase was also identified in the deduced amino acid sequence. It has been proposed  that for Type 1 copper ligand, residues located 10 amino acids downstream of the conserved Cys affect the redox potential and this provides a basis for classification of laccase under class 1 (Met), class 2 (Leu) or class 3(Phe). The sequence LEA adjacent to the last conserved His is conserved in laccases of high redox potential with Ala at the most being replaced by other residues. This contrasts with the laccases of low redox potential which have a sequence of VSG replacing the LEA tripeptide. The presence of Leu at the Type 1 copper binding position and LEL tripeptide (Figure 1, last row, red box #2) suggests the C. bulleri laccase to be a high redox potential enzyme. Moreover, the presence of Glu 460 and Ser 113 at the equivalent conserved positions further strengthens this hypothesis. The sequence also showed the presence of pre-sequence in agreement with its extracellular localization. The sequence conservation in this region between various fungi was observed to be poor indicating the use of different secretion peptides in different fungal species.
The C. bulleri laccase was expressed at a level of 600–720 U L-1 using pPICZαB yeast shuttle vector. In general, low heterologous expression of fungal laccase has been reported in P. pastoris (less than 1000 U L-1) compared to the expression levels in the native fungi. Enhancement in extracellular laccase activity has been reported by addition of copper sulfate [7, 9] to the culture medium of P. pastoris. Mutagenesis of the structural gene followed by expression in P. pastoris also lead to enhanced expression of Trametes sp. AH28 -2 laccase . In the present work, optimization of the time of addition and concentration of copper salt lead to an increase in laccase activity to ~7200 U L-1 in 6 days. While this effect of copper has been reported at the level of transcription, mediated by copper-dependent responsive element in some fungi , no such effect is expected in the recombinant P. pastoris as the laccase gene is transcribed under the control of the AOX1 promoter. Addition of copper salts in the medium does not affect extracellular laccase activity in the native C. bulleri either (unpublished data). Since laccases are metallo-proteins, it is likely that the addition of copper allows the excreted laccase in P. pastoris to fold correctly in the culture filtrate.
The rLac appeared as a 60 ± 5 kDa protein, slightly higher and more heterogeneous when compared to the nLac (58 ± 5 kDa)  and these differences were attributed to increased glycosylation of the rLac in P. pastoris (see below). While the kinetic parameters against ABTS, guaiacol and pyrogallol were found to be similar (Additional file 2: Table S1) for the native and the rLac, the latter was found to be more thermo-stable. This thermo-stability is likely to be on account of higher glycosylation in P. pastoris. It has been proposed that the glycans, being highly hydrophilic in nature, contribute to the stability by associating covalently to the amino acid residues present on the surface of the protein molecules . The higher stability is expressed by higher melting temperature. The recent work on the engineered SH3 domain variants also clearly suggested that glycosylation can enrich as well as modulate the biophysical properties of proteins and could, in fact, be used as an alternative way to design thermally stabilized proteins . In this study, we can also correlate the tolerance to organic solvents as a by-product of this altered glycosylation pattern. As observed, the rLac produced in P. pastoris exhibited higher tolerance towards various water-miscible organic solvents compared to the native laccases from T. versicolor and Pleurotus ostreatus. Between 40-60% residual activity was observed at 50% (v/v) in all these solvents after 3 h of incubation. While these values were slightly lower than those observed for the nLac (Figure 4), these are still high and useful for its use in organic synthesis work. Interestingly, both the rLac and the nLac were equally unstable in THF (solvent of a higher log P value)which is likely to have distorted the enzyme hydration and distort the conformation leading to a drastic decrease in enzyme activity. It has been observed that laccase structure, stability and activity are affected by water miscible solvents through direct interaction with enzyme and through its affect on water activity (aw) . Although Farnet et al.  have observed a high IC50 values (30-60%) of the Marasmius quercophilus laccase in different solvents but the enzyme was not incubated for longer time periods and thus their data cannot be compared to our results.
Laccases are generally inhibited by chloride ions, an important component in dye wastewaters, which limits its use in treatment plants. Chloride ions directly affect the conversion process through their intrinsic effects on rate constants mediated through availability of Type 2 and Type 3 copper atoms in the active site . Higher resistance to chloride ions (after 2 h incubation) was observed for the rLac of C. bulleri (Figure 5) when compared to the nLac. A chloride tolerant laccase having IC50 of 1.5 M was recently reported  but again, the enzyme was not incubated for long time periods and hence cannot be compared to the laccase expressed in this study.
For many of the differences observed between the rLac and the nLac, a detailed comparison of the trypsin digested peptide fragments was made. Several peptides were found to be identical confirming the expression of the same laccase in P. pastoris, as reported previously from our group. Out of the 4 putative glycosylation sites, only 2 were likely to get glycosylated . Differences in the glycosylation patterns, leading to generation of a spectrum of different peptides, were observed. Software tools were used to identify these and the fragment with m/z of 2593.9521 (obtained from the rLac) was concluded to represent the aa sequence 442–456 with possible glycan structure of (Hex)4 (HexNAc)2 (Sulph/Phos)1. The corresponding fragment from the nLac was identified at 2132.1 m/z with an assigned structure of (Hex)1(HexNAc)2(NeuGc)1. While theoretically, additional peptide (28 VISPDGFNRSAVLAGGTADNADFPGPLVTGNK 38) is predicted to undergo glycosylation and may indeed do so, this is not likely to be detected by MALDI-TOF MS, as the size of this exceeds the detection limits of the system.