Molecular cloning of a laccase gene from Ganoderma lucidum and heterologous expression in Pichia pastoris

Abstract

A genomic laccase gene and cDNA were cloned from the white-rot fungi Ganoderma lucidum TR6. The genomic laccase gene contained 2086 bp with nine introns. The laccase cDNA had an open reading frame of 1563 bp. The deduced mature protein consisted of 520 amino acids. Both the genomic laccase gene and cDNA were expressed in the Pichia pastoris GS115. Laccase activities could be detected in transformants with laccase cDNA but not in transformants with genomic laccase gene. The highest activity value reached 685.8 U L(-1). The effects of temperature, pH and nitrogen source on laccase expression in P. pastoris were analyzed. The recombinant laccase was purified and the molecular mass was 73.4 KDa, a little bigger than native laccase. The optimal pH and temperature were specific at pH 3.5 and special range from 60 to 90 °C. The laccase was stable at pH 7.0 and temperature range of 20-30 °C. The Km and Vm values of this recombinant laccase for ABTS were 0.521 mM and 19.65 mM min(-1), respectively.