Periplasmic expression of Pseudomonas fluorescens peroxidase Dyp1B and site-directed mutant Dyp1B enzymes enhances polymeric lignin degradation activity in Pseudomonas putida KT2440

Expression of lignin-oxidising Pseudomonas fluorescens Dyp1B in the periplasm of Pseudomonas putida KT2440, using a tat fusion construct, was found to lead to enhanced whole cell activity for oxidation of DCP and polymeric lignin substrates. Four amino acid residues predicted to lie at the manganese ion binding site of Pseudomonas fluorescens peroxidase Dyp1B were investigated using site-directed mutagenesis. Mutants H127R and S223A showed 2-fold and 4-fold higher kcat for Mn(II) oxidation respectively, and mutant S223A showed 2-fold enhanced production of low molecular weight phenolic products from a polymeric soda lignin. The mutant Pfl Dyp1B genes were expressed as tat fusions to investigate their effect on lignin oxidation by P. putida KT2440.