Abstract
The cDNA encoding a major laccase isozyme (Lac1) produced by Grifola frondosa in culture fluid was cloned. The lac1 cDNA encodes a mature Lac1 of 499 amino acid residues, preceded by a signal peptide with 21 amino acid residues. The lac1 cDNA was expressed in Pichia pastoris. The recombinant Lac1 (rLac1) was produced as a secreted protein with its own signal peptide. The addition of CuSO4 to the culture medium was effective in increasing laccase activity. The molecular mass of purified rLac1 was estimated to be 95 kDa, which is greater than that of the native form, suggesting that rLac1 is highly glycosylated. The catalytic efficiency (kcat/Km) of rLac1 was lower than that of native Lac1 (nLac1) for all substrates tested. However, rLac1 exhibited higher thermostability and stability in acidic conditions compared to nLac1. In addition, rLac1 showed a high capacity for decolorization of several synthetic dyes comparable to nLac1. These results demonstrated the higher application potential of rLac1 than that of nLac1.
