Protein Engineering of Dyp1B from Pseudomonas fluorescens Pf-5 for Improved Manganese and Lignin Oxidation

  • Austine Ehibhatiomhan

Student thesis: Doctoral ThesisDoctor of Philosophy


Lignin is an organic aromatic polymer which constitutes 15%-30% of most plant biomass as well as being one of the largest by-product of 2nd generation biofuel production and paper/pulp manufacture. Delignification processes for lignocellulosic biomass have gathered attention for its commercial and environmental benefits like the production of organic chemicals and hydrocarbon polymers i.e., bioethanol, guaiacol, vanillin and ferulic acid. Micro-organisms namely Fungi and bacterial produce extracellular lignolytic peroxidases i.e., manganese peroxidases (MnP), lignin peroxidases (LiP), laccases (Lac), and versatile peroxidases (VP). Fungi peroxidases produced by the white-rot fungi P. chrysosporium have been extensively characterised for lignin oxidation however, bacterial peroxidases have recently amassed more consideration due to favourable industrial characteristics like mutagenesis studies for improved activities and specific reaction conditions i.e., thermostability. A novel bacterial peroxidase, Dye-decolorising peroxidase (Dyp), catalyses the peroxide-dependent oxidation of divalent manganese (Mn2+) as seen with fungi MnPs, some Dyps have shown lignin oxidation capabilities with improved lignin oxidation observed in the presence of Mn2+. Mutagenesis studies have also been carried out on Dyps for improved lignin oxidation, a Dyp homologue from R.jostii RHA1 has been previously shown to display improved Mn2+ and subsequently improved lignin oxidation via mutagenesis studies of amino acid residues involved in Mn2+ oxidation (Ramachandra, Crawford, and Hertel 1988, Roberts et al. 2011, Singh et al. 2013). Herein, this PhD aimed to add to existing knowledge on the role bacterial Dyps and their mediators play in the degradation of lignin. Dyp1B from the Gram-negative Pseudomonas fluorescens Pf-5 was investigated for improved Mn2+ and lignin oxidation by investigation of amino acids predicted to be implicated in the binding/activity of Mn2+. The role of secondary coordination sphere interactions within the active site of PfDyp1B and their implications on activity towards Mn2+ was explored as well as the relationship between Mn2+ binding/activity and lignin oxidation. The resulting mutants were expressed as Apo-proteins, purified, profiled for pH and thermo stability, and characterised for peroxidase and ligninolytic activities. Kinetic parameters were measured and analysed. Some of the resulting mutants displayed improved catalytic efficiency towards Mn2+ with the PfDyp1B variant S223A displaying a kcat/KM value of 3.1x104 M-1s-1, 8-fold higher than the wild type variant. Improved release of low molecular weight (LMW) products was also observed for a number of mutants and through reverse phase High-performance liquid chromatography (HPLC) improved release of vanillin was detected for one of the PfDyp1B variants. The In vivo expression of the PfDyp1B variants highlighted the potential for P. putida KT2440 to be utilised as a biotechnological cell factory for the valorisation of lignin.
Date of AwardSept 2022
Original languageEnglish
Awarding Institution
  • Coventry University
SupervisorSebastien Farnaud (Supervisor), Sharon Williams (Supervisor) & Timothy D H Bugg (Supervisor)

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