Expression in yeast of secreted lignin peroxidase with improved 2,4-dichlorophenol degradability by DNA shuffling

Abstract

Lignin peroxidase (LiP) from Phanerochaete chrysosporium was shown to mineralize a variety of recalcitrant aromatic compounds and oxidize a number of polycyclic aromatic and phenolic compounds. The major problem of the wild type LiP is that it can be inactivated by excess H2O2 and high concentrations of aromatic compounds. We applied a directed evolution technique coupled with a rapid colorimetric screening method to obtain mutant genes with improved H2O2 stability and polychlorinated phenol degradability, and they were successfully expressed as the secretive LiPs in recombinant Saccharomyces cerevisiae. The resulting variants showed approximately 1.6-fold improved 2,4-dichlorophenol (2,4-DCP) degradation activity and stability against H2O2 compared with the parent strain. The kinetic properties of the variants toward 2,4-DCP and H2O2 were also increased compared with the wild type for all three mutants studied. Amino acid sequence analysis indicated that the greatest number of amino acid substitutions was located near the surface or Ca2+ binding sites of the enzyme.