Our group has sequenced the genome of L. edodes L54 using Roche 454 pyrosequencing. GeneMarkES in silco predicted protein-coding genes based on the genome sequence. Alignment with FOLy revealed 14 putative laccase genes. Reverse transcription polymerase chain reaction showed that ten of them (L1-L10) actively expressed in the mycelial stage. Laccase L4 clones yielded two allelic forms with 21 single nucleotide polymorphisms and five amino acid substitutions. Both alleles were cloned and expressed in Pichia pastoris and purified. Comprehensive kinetic and stability analyses suggested similar properties between the alleles. Most importantly, both recombinant enzymes demonstrated potent catalysis in decolourisation of 8 synthetic dyes and in degradation of polyaromatic hydrocarbons. In conclusion, this study provides a platform for engineering L. edodes laccases, and two versatile recombinant enzymes for “green” applications.
P49: Pichia pastoris expresses functional Lentinula edodes laccases: Bridging genome to “green” applications
Sunday, July 24, 2011
Grand Ballroom, 5th fl (Sheraton New Orleans)
The Shiitake mushroom, Lentinula edodes, is the second most cultivated edible mushroom. It belongs to white-rot basidiomycete that can degrade lignocellulosic materials with enzymes such as laccases. Laccases are multi-copper oxidases that carry out oxidative ligninolysis. The enzymes have high industrial importance with diverse applications including pulp delignification, bioremediation and bioethanol production. Their high application versatility has led to high demands. However, the sources of laccases are few. Extraction of laccases from fungi such as L. edodes are tedious and expensive for industrial applications. Cloning and heterologous expression of L. edodes laccases could be a cost-effective alternative for enzyme preparations.