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

doi:10.1016/j.enzmictec.2019.01.002

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

School of Life Sciences

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

30 Citations (Scopus)

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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.

Original language	English
Pages (from-to)	21-29
Number of pages	9
Journal	Enzyme and Microbial Technology
Volume	123
Early online date	9 Jan 2019
DOIs	https://doi.org/10.1016/j.enzmictec.2019.01.002
Publication status	Published - Apr 2019

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

Access to Document

10.1016/j.enzmictec.2019.01.002

Post-PrintAccepted author manuscript, 1.42 MBLicence: CC BY-NC-ND

Cite this

@article{42345daacc4841d5b776142fde85b4e0,

title = "Protein engineering of Pseudomonas fluorescens peroxidase Dyp1B for oxidation of phenolic and polymeric lignin substrates",

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.",

keywords = "Directed evolution, Dye decolorizing peroxidase, Lignin bioconversion, Protein engineering",

author = "Pour, {Rahman Rahman} and Austine Ehibhatiomhan and Yuling Huang and Ben Ashley and Goran Rashid and {Mendel Williams}, Sharon and Bugg, {Timothy D H}",

note = "NOTICE: this is the author{\textquoteright}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 {\textcopyright} 2019, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ ",

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month = apr,

doi = "10.1016/j.enzmictec.2019.01.002",

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T1 - Protein engineering of Pseudomonas fluorescens peroxidase Dyp1B for oxidation of phenolic and polymeric lignin substrates

AU - Pour, Rahman Rahman

AU - Ehibhatiomhan, Austine

AU - Huang, Yuling

AU - Ashley, Ben

AU - Rashid, Goran

AU - Mendel Williams, Sharon

AU - Bugg, Timothy D H

N1 - 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/

PY - 2019/4

Y1 - 2019/4

N2 - 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.

AB - 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.

KW - Directed evolution

KW - Dye decolorizing peroxidase

KW - Lignin bioconversion

KW - Protein engineering

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DO - 10.1016/j.enzmictec.2019.01.002

M3 - Article

SN - 1879-0909

VL - 123

SP - 21

EP - 29

JO - Enzyme and Microbial Technology

JF - Enzyme and Microbial Technology

ER -

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

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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Sharon Williams

Cite this

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

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Profiles

Sharon Williams

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