SCAN:Integrating Protein Science and Protein Technology

SCAN Seminar - ITQB

Title: Integrating Protein Science and Protein Technology

Speaker: Lígia O. Martins

affiliation: Microbial & Enzyme Technology Laboratory

Abstract:

Modern biocatalysis is achieving new advances in environmental fields, from enzymatic bioremediation to the synthesis of renewable and clean energies. From an environmental point of view, the use of enzymes instead of chemicals or microorganisms presents several advantages including the potential for their production at a higher scale, with enhanced stability and/or activity and at a lower cost by using recombinant-DNA technology.
Multicopper oxidases (that includes laccases and metallo-oxidases) are versatile enzymes capable of coupling the oxidation of a variety of substrates namely phenols, diamines and even some inorganic compounds, to the reduction of dioxygen. These enzymes are ubiquitously present in all Domains of Life. Laccases in particular, are very interesting enzymes for a range of biotechnological applications due to the lack of expensive cofactors requirement and the use of readily available oxygen as electron acceptor.
In the last years, we become interested in exploring the functional and structural features of multicopper oxidases from prokaryotes for which genetic tools and biotechnological processes are well established. Our research focused on the CotA-laccase of Bacillus subtilis and, on the metalloxidases McoA and McoP from Aquifex aeolicus and Pyrobaculum aerophilum, respectively. These are thermoactive and thermostable proteins with Top and Tm values above 80°C. Several interesting features of the enzymes were unravelled such as the putative role of McoA in the organism mechanism of copper homeostasis or the involvement of McoP in the last step of the denitrification pathway. The key role of copper in the activity and conformational stability of these enzymes has been addressed. The characterization of mutants obtained by site-directed mutagenesis contributed for the clarification of the catalytic mechanism of these enzymes, namely the factors that modulate the redox potential of the T1 copper site and the reduction of dioxygen at the T2/T3 copper cluster. In addition, directed evolution methodologies by error-prone PCR and saturation mutagenesis followed by high-throughput screening have been pursued in order to find enhanced biocatalysts towards e.g. organic solvent denaturation. Finally, CotA-laccase was tested on its capacity to degrade synthetic dyes. These are anthropogenic pollutants increasingly being used in the food, pharmaceutical, textile and leather industries. Over 100,000 commercially dyes exist and more than 7x105 tons of dyestuff is produced annually of which 1-1.5x105 is released to the environment in wastewaters, especially from the textile industry. Combining enzymology, electrochemistry, mass spectrometry and nuclear magnetic resonance, a detailed characterization of the laccase decolourisation system was achieved and mechanistic pathways for azoic and antraquinonic dyes conversion were proposed. This will guide us to further develop efficient biodegradation treatment technologies by using protein engineering tools.