Biocatalyst Characterization

We have undertaken a multidisciplinary study of CotA-laccase from Bacillus subtilis as a model bacterial laccase system. Fundamental studies were aimed at clarifying the catalytic activity and stability of this enzyme. X-ray studies in collaboration with Protein Modelling Lab and Crystallographic Unit have given structural insight into the nature of the catalytic centres, the interaction of reduced substrates with the binding site and into the principal stages of the mechanism of dioxygen reduction. Site directed mutagenesis was used to examine how the replacement of key residues affected the structure, the spectroscopic properties, the redox potential and catalytic properties of variant enzymes. In particular we have addressed 1) the effect of solvent accessibility of the T1 Cu centre and the electrostatic interactions between this centre and the protein with effect in the redox potential of the enzyme (Durão et al. 2006, 2008), 2) the thermodynamic stability of CotA as well as of McoA and the effect of removal of the disulfide bridge in the enzyme activity and stability (Durão et al. 2006, Fernandes et al. 2011), 3) the mechanism of dioxygen reduction and the role of negatively charged residues close to the trinuclear copper center in protonation events (Chen et al. 2010, Bento et al. 2010, Silva et al. 2012, Brissos et al. 2012).

Recently, in collaboration with Raman Spectroscopy of Metalloproteins Laboratory Dye-decolourizing peroxidase PpDyP from Pseudomonas putida  MET94 was successfully immobilized in Ag electrodes electrodes coated with alkanethiol self assembled monolayer. In the immobilized state the enzyme (i) the structural integrity of the heme pocket is preserved, (ii) the enzyme is electronically coupled to the electrode and (iii) it exhibits efficient catalytic activity showing that the enzyme is a promising candidate for the design of novel bio-electronic devices (Sezer et al. 2012). Relevant spectroscopic insights into structural details into the redox properties of PpDyP and BsDyP from Bacillus subtilis was obtained (Sezer et al.

 

MET | Microbial & Enzyme Technology 2013