Abstract
BackgroundDye-decolorizing peroxidases (DyPs) are haem-containing peroxidases that show great promises in industrial biocatalysis and lignocellulosic degradation. Through the use of Escherichia coli osmotically-inducible protein Y (OsmY) as a bacterial extracellular protein secretion system (BENNY), we successfully developed a streamlined directed evolution workflow to accelerate the protein engineering of DyP4 from Pleurotus ostreatus strain PC15.ResultAfter 3 rounds of random mutagenesis with error-prone polymerase chain reaction (epPCR) and 1 round of saturation mutagenesis, we obtained 4D4 variant (I56V, K109R, N227S and N312S) that displays multiple desirable phenotypes, including higher protein yield and secretion, higher specific activity (2.7-fold improvement in kcat/Km) and higher H2O2 tolerance (sevenfold improvement based on IC50).ConclusionTo our best knowledge, this is the first report of applying OsmY to simplify the directed evolution workflow and to direct the extracellular secretion of a haem protein such as DyP4.
Highlights
Dye-decolorizing peroxidases (DyPs; PF04261; EC 1.11.1.19) comprise a recently described family of haem peroxidase enzymes, which is unrelated to the superfamilies of plant and animal peroxidases (Martinez et al 2017)
For DyP4, the optimum H2O2 concentration for ABTS oxidation was determined to be 0.25 mM. This was consistent with a previous study that used exactly the same H2O2 concentration for the oxidation of ABTS, M n2+, Reactive Blue 19 (RB19), Reactive Black 5 (RB5) and 2,6-dimethoxyphenol (DMP) (Fernandez-Fueyo et al 2015)
0.2 mM H 2O2 was used for ABTS, guaiacol and DMP oxidation by bacterial dyedecolorizing peroxidase (DyP) from Pseudomonas putida MET94 (PpDyP) (Brissos et al 2017)
Summary
Dye-decolorizing peroxidases (DyPs; PF04261; EC 1.11.1.19) comprise a recently described family of haem peroxidase enzymes, which is unrelated to the superfamilies of plant and animal peroxidases (Martinez et al 2017). According to the RedoxiBase [http://peroxibase.toulouse.inra.fr/; accessed on 07/04/19, (Fawal et al 2013)] a total of 237 DyPs have been identified in the genomes of fungi, bacteria and archaea. Their physiological functions are yet to be fully elucidated, DyPs have several characteristics that distinguish them from all other peroxidases. Accumulating evidence shows that DyPs play a key role in lignin degradation. Owing to these unique properties, DyPs are potential candidates for a variety of biotechnological applications. Dye-decolorizing peroxidases (DyPs) are haem-containing peroxidases that show great promises in industrial biocatalysis and lignocellulosic degradation. Through the use of Escherichia coli osmotically-inducible pro‐ tein Y (OsmY) as a bacterial extracellular protein secretion system (BENNY), we successfully developed a streamlined directed evolution workflow to accelerate the protein engineering of DyP4 from Pleurotus ostreatus strain PC15
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