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

doi:10.1016/j.enzmictec.2022.110147

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

School of Life Sciences

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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 k_cat 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 language	English
Article number	110147
Number of pages	8
Journal	Enzyme and Microbial Technology
Volume	162
Early online date	20 Oct 2022
DOIs	https://doi.org/10.1016/j.enzmictec.2022.110147
Publication status	Published - Jan 2023

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

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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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Ehibhatiomhan, A. O., Pour, R. R., Farnaud, S., Bugg, T. D. H., & Mendel-Williams, S. (2023). Periplasmic expression of Pseudomonas fluorescens peroxidase Dyp1B and site-directed mutant Dyp1B enzymes enhances polymeric lignin degradation activity in Pseudomonas putida KT2440. Enzyme and Microbial Technology, 162, [110147]. https://doi.org/10.1016/j.enzmictec.2022.110147

Periplasmic expression of Pseudomonas fluorescens peroxidase Dyp1B and site-directed mutant Dyp1B enzymes enhances polymeric lignin degradation activity in Pseudomonas putida KT2440. / Ehibhatiomhan, Austine O.; Pour, Rahman Rahman ; Farnaud, Sebastien; Bugg, Timothy D.H,; Mendel-Williams, Sharon.

In: Enzyme and Microbial Technology, Vol. 162, 110147, 01.2023.

Research output: Contribution to journal › Article › peer-review

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

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.",

keywords = "Dye-decolorizing peroxidases, Lignin degradation, Manganese oxidation, Pseudomonas fluorescens, Pseudomonas putida",

author = "Ehibhatiomhan, {Austine O.} and Pour, {Rahman Rahman} and Sebastien Farnaud and Bugg, {Timothy D.H,} and Sharon Mendel-Williams",

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AU - Ehibhatiomhan, Austine O.

AU - Pour, Rahman Rahman

AU - Farnaud, Sebastien

AU - Bugg, Timothy D.H,

AU - Mendel-Williams, Sharon

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AB - 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.

KW - Dye-decolorizing peroxidases

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KW - Manganese oxidation

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

Abstract

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