RESUMO
Copper ion was necessary for the transcription of all laccase isozyme genes from Trametes sp. AH28-2, with higher concentrations of Cu2+ (1-2 mmol/L) being more favorable to the synthesis of laccase. In the glucose media containing 0.5 mmol/L Cu2+, the laccase activity of the supernate was rather low (44.3 u/L) and had an increase of 60.3% (71.0 u/L) when 4.0 mmol/L o-toluidine was added. Moreover, the activity reached up to 2584 u/L as the glucose was replaced by cellobiose. And Native-PAGE showed that LacA was the main laccase component if fungus was induced by o-toluidine or copper ions. It had been demonstrated by quantitative RT-PCR that the regulation of lacA expression, induced by o-toluidine, occurred at the transcriptional level, with the accumulation of mRNA transcripts being accompanied by the increase of laccase activity of the culture fluid. In addition, the structural gene of lacA interrupted by 10 introns was 2110 bp in length and the corresponding cDNA sequence was 1560 bp encoding a 520 aa protein, which had high similarities with other laccases from basidiomycetes. Furthermore, a length of 1881 bp of 5'-terminal sequence of transcription control of lacA, amplified by the improved inverse PCR, contained a TATA box, seven CAAT boxes as well as a number of putative cis-acting elements important for its expression, including five MREs, nine CreA-binding sites, four XREs, two STREs and seven nitrogen factor binding sites. The existence of these elements was well in agreement with the data obtained from Trametes sp. AH28-2 shaken-flask cultures.