Protein engineering of Pseudomonas fluorescens peroxidase Dyp1B for oxidation of phenolic and polymeric lignin substrates

Rahman Rahman Pour, Austine Ehibhatiomhan, Yuling Huang, Ben Ashley, Goran Rashid, Sharon Mendel Williams, Timothy D H Bugg

Research output: Contribution to journalArticle

Abstract

Directed evolution was applied to dye-decolourizing peroxidase Dyp1B from Pseudomonas fluorescens Pf-5, in order to enhance the activity for oxidation of phenolic and lignin substrates. Saturation mutagenesis was used to generate focused libraries at 7 active site residues in the vicinity of the heme cofactor, and the libraries were screened for activity towards 2,6-dichlorophenol. Mutants N193 L and H169 L were found to show 7–8 fold enhanced kcat/KM towards DCP, and replacements at Val205 and Ala209 also showed enhanced activity towards alkali Kraft lignin. Residues near the predicted Mn(II) binding site were also investigated by site-directed mutagenesis, and mutants S223 N and H127R showed 4-7-fold increased kcat/KM for Mn(II) oxidation. Mutant F128R also showed enhanced thermostability, compared to wild-type Dyp1B. Testing of mutants for low molecular weight product release from Protobind alkali lignin revealed that mutant H169 L showed enhanced product release, compared with WT enzyme, and the formation of three low molecular weight metabolites by this mutant was detected by reverse phase HPLC analysis.
LanguageEnglish
Pages21-29
Number of pages9
JournalEnzyme and Microbial Technology
Volume123
Early online date9 Jan 2019
DOIs
Publication statusPublished - Apr 2019

Fingerprint

protein engineering
Pseudomonas fluorescens
lignin
peroxidase
oxidation
mutants
alkalis
molecular weight
site-directed mutagenesis
heme
thermal stability
mutagenesis
active sites
dyes
binding sites
metabolites
enzymes

Bibliographical note

NOTICE: this is the author’s version of a work that was accepted for publication in Enzyme and Microbial Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Enzyme and Microbial Technology, [[Vol 123] (2019)] DOI: 10.1016/j.enzmictec.2019.01.002

© 2019, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/

Keywords

  • Directed evolution
  • Dye decolorizing peroxidase
  • Lignin bioconversion
  • Protein engineering

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology

Cite this

Protein engineering of Pseudomonas fluorescens peroxidase Dyp1B for oxidation of phenolic and polymeric lignin substrates. / Pour, Rahman Rahman ; Ehibhatiomhan, Austine; Huang, Yuling; Ashley, Ben; Rashid, Goran; Mendel Williams, Sharon; Bugg, Timothy D H.

In: Enzyme and Microbial Technology, Vol. 123, 04.2019, p. 21-29.

Research output: Contribution to journalArticle

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