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 N193L and H169L 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 S223N 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 H169L 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.
Submitter: Timothy Bugg
Submission Date: Jan. 17, 2019, 12:57 a.m.
|Number of data points||108|
|Proteins||Dye-decolorizing peroxidase Dyp1B|
|Assays/Quantities/Protocols||Experimental Assay: 2,4-dichlorophenol: kcat/Km ; Experimental Assay: 2,4-dichlorophenol: kcat ; Experimental Assay: Mn2+ oxidation: kcat/Km ; Experimental Assay: 2,4-dichlorophenol: Km ; Experimental Assay: Mn2+ oxidation: kcat ; Experimental Assay: Mn2+ oxidation: Km ; Derived Quantity: SD of 2,4-dichlorophenol: kcat ; Derived Quantity: SD of 2,4-dichlorophenol: kcat/Km ; Derived Quantity: SD of 2,4-dichlorophenol: Km ; Derived Quantity: SD of Mn2+ oxidation: kcat/Km ; Derived Quantity: SD of Mn2+ oxidation: kcat ; Derived Quantity: SD of Mn2+ oxidation: Km|
|Libraries||Table 2: Kinetic parameters from directed evolution ; Table 3: Kinetic parameters for site-directed mutants at Mn(II) binding site|