1 Department of Chemistry, Technical University of Denmark2 Department of Chemical and Biochemical Engineering, Technical University of Denmark3 Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark4 Physical and Biophysical Chemistry, Department of Chemistry, Technical University of Denmark
This paper reports the identification, heterologous expression in Escherichia coli and characterization of TtMCO from the thermophilic bacterium Thermobaculum terrenum, the first laccase-like multi-copper oxidase (LMCO) from the distinct Phylum Chloroflexi. TtMCO has only 39% identity to its closest characterized homologue, CotA from Bacillus subtilis, but sequence and spectrophotometry confirmed copper coordination similar to that of LMCOs. TtMCO is extremely thermophilic with a half-time of inactivation of 2.24 days at 70 degrees C and 350 min at 80°C and pH 7, consistent with a hyperthermal habitat of the host. TtMCO was screened for activity against 56 chemically diverse substrates. It displayed limited activity on classical LMCO substrates, such as e.g. phenolics, transition metals, or bilirubin. Highest activities were observed for nitrogen-containing aromatic compounds, i.e. 1,8-diaminonaphtalene (Km = 0.159 mM, kcat = 0.295 s-1) and ABTS (Km = 0.844mM, kcat= 2.13 s-1). The combined data suggest a distinct role of TtMCO and a substantial trade-off between activity and stability, compared to other characterized bacterial LMCOs, making it of interest in future protein engineering studies.
Journal of Molecular Catalysis B: Enzymatic, 2015, Vol 112, p. 59-65