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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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WRAP-protein-engineering-Pseudomonas-fluorescens-peroxidase-Dyp1B-Bugg-2019.pdf - Accepted Version - Requires a PDF viewer. Available under License Creative Commons Attribution Non-commercial No Derivatives 4.0. Download (1508Kb) | Preview |
Official URL: http://dx.doi.org/10.1016/j.enzmictec.2019.01.002
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 | |||||||||
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| 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: |
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| 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: |
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