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

Austine O. Ehibhatiomhan, Rahman Rahman Pour, Sebastien Farnaud, Timothy D.H, Bugg, Sharon Mendel-Williams

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Abstract

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.

Original languageEnglish
Article number110147
Number of pages8
JournalEnzyme and Microbial Technology
Volume162
Early online date20 Oct 2022
DOIs
Publication statusPublished - Jan 2023

Bibliographical note

This is an open access article under the CC-BY license,

Funder

Funding Information:
This work was supported by BBSRC ERA-IB research grant BB/M025772/1 , and a Ph.D. studentship from Coventry University (to A.O.E.). We would like to thank Dr. Eduardo Díaz (CSIC Madrid) for the gift of plasmid pIZ1016, Dr. James Williamson (University of Warwick) for practical advice and assistance, and Dr Goran Rashid (University of Warwick) for assistance with HPLC.

Funding Information:
This work was supported by BBSRC ERA-IB research grant

Keywords

  • Dye-decolorizing peroxidases
  • Lignin degradation
  • Manganese oxidation
  • Pseudomonas fluorescens
  • Pseudomonas putida

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology

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