Trametes versicolor laccase: random mutagenesis and heterologous expression in Pichia pastoris

Laccase is a blue multi-copper oxidase. It has a broad biotechnical potential which increases the interest to study the enzyme further. A laccase-encoding gene from the white-rot fungus Trametes versicolor (lcc2) was mutated using two different methods for random mutagenesis: error-prone PCR and a method based on an E. coli strain (ES1301 mutS) that introduces random mutations. For the error-prone PCR reaction, the vector pPICZB with the lcc2 gene inserted was used as template. The E. coli strain ES1301 mutS was transformed with the vector pBluescript SKII with the lcc2 gene as insert. The mutagenesis products were cloned into the Pichia pastoris expression vector pPICZB for transformation of P. pastoris SMD1168. The transformants were spread on agar plates containing zeocin. Laccasesecreting transformants were selected by their ability to oxidize the substrates ABTS [2,2 ́azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] and syringaldazine [N,N ́-bis(3,5dimethoxy-4-hydroxybenzylidene)hydrazine], the products of which give green and purple colour, respectively. Around 20 transformants from each of the mutagenesis methods were transferred to plates containing 1 mM ABTS or 1 mM syringaldazine. None of the transformants produced any colour. Control transformants (pPICZB with unmutated lcc2) were also spread on plates with either ABTS or syringaldazine. The transformants gave rise to green colour after 24 hours on the ABTS plates and to purple colour after 72 hours on the syringaldazine plates. Experiments with different chromogenic substrates indicated that ABTS and syringaldazine were best suited for screening of mutants. Remazol Brilliant Blue and Phenol Red are two substrates that after optimisation can serve as alternatives for the selection of laccase-secreting transformants.