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

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Rahmanpour, Rahman, Ehibhatiomhan, Austine, Huang, Yuling, Ashley, Ben, Rashid, Goran M. M., Mendel-Williams, Sharon and Bugg, Timothy D. H. (2019) Protein engineering of pseudomonas fluorescens peroxidase Dyp1B for oxidation of phenolic and polymeric lignin substrates. Enzyme and Microbial Technology, 123 . pp. 21-29. doi:10.1016/j.enzmictec.2019.01.002

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Official URL: http://dx.doi.org/10.1016/j.enzmictec.2019.01.002

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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.

Item Type: Journal Article
Subjects: T Technology > TP Chemical technology
Divisions: Faculty of Science > Chemistry
Library of Congress Subject Headings (LCSH): Protein engineering, Lignin
Journal or Publication Title: Enzyme and Microbial Technology
Publisher: Elsevier Inc.
ISSN: 0141-0229
Official Date: April 2019
Dates:
DateEvent
April 2019Published
9 January 2019Available
4 January 2019Accepted
Volume: 123
Page Range: pp. 21-29
DOI: 10.1016/j.enzmictec.2019.01.002
Status: Peer Reviewed
Publication Status: Published
Access rights to Published version: Restricted or Subscription Access
RIOXX Funder/Project Grant:
Project/Grant IDRIOXX Funder NameFunder ID
BB/M025772/1[BBSRC] Biotechnology and Biological Sciences Research Councilhttp://dx.doi.org/10.13039/501100000268
720303H2020 European Research Councilhttp://dx.doi.org/10.13039/100010663

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