Synthesis and application of a phenazine class substrate for high-throughput screening of laccase activity.

Biocatalysis is one of the greatest tools for implementing the 12 principles of Green chemistry. Biocatalysts are bio-based, highly efficient and selective, operate at moderate conditions, and can be reused multiple times. However, the wider application of biocatalysts is plagued by a plethora of drawbacks, such as poor stability at operating conditions, inadequate efficiency of catalytic systems, a small number of commercially available biocatalysts, and a lack of substrates or methods for their discovery and development. In this work, we address the lack of suitable substrates for high-throughput screening of laccase by synthesising and investigating a newly developed phenazine-type substrate - Ferbamine. Investigation of Ferbamine pH and thermal stability indicated that its long-term stability in an aqueous medium is superior to that of commercially available substrates and does not require organic solvents. Ferbamine displayed convincing performance in detecting laccase activity on Ferbamine-agar plates in commercial laccase products and the collection of extracts from wild terrestrial fungi (42 species, 65 extracts), of which 26 species have not been described to have laccase activity prior to this work. Incubation of microorganisms on Ferbamine-agar plates showed its compatibility with live colonies. Ferbamine proved to be an easy-to-use substrate, which could be a great addition to the toolbox of methods for the functional analysis of laccases.