Functional expression of Trametes versicolor thermotolerant laccase variant in Pichia pastoris

A mutational laccase gene MLcc1 was synthesized with modified codons, based on the codon bias of Pichia pastoris, in order to improve the thermal stability of Trametes versicolor laccase. The mutant gene was subcloned into the expression vector pGAPZαA and was transformed into P. pastoris strain X33. The mature protein consisted of 498 amino acids and contained a changed aspartate with proline at the 14th position of the native laccase (NLCC1). Under optimum conditions, the laccase activity reached 194.06 U/L after 54 h by high cell density fermentation in a 5 L fermenter. After incubation at pH 4.6 for 2 h, the recombinant enzyme MLCC1 retained 54.9% of its maximum activity, whereas the wild-type enzyme retained about 50% of its maximum activity. More than 20% of the residual activity was detected after up to 120 min at 90 °C for MLCC1, whereas less than 10% of the activity was retained for NLCC1.