Bilirubin oxidase from Bacillus pumilus: A promising enzyme for the elaboration of efficient cathodes in biofuel cells

A CotA multicopper oxidase (MCO) from Bacillus pumilus, previously identified as a laccase, has been studied and characterized as a new bacterial bilirubin oxidase (BOD). The 59 kDa protein containing four coppers, was successfully over-expressed in Escherichia coli and purified to homogeneity in one step. This 509 amino-acid enzyme, having 67% and 26% sequence identity with CotA from Bacillus subtilis and BOD from Myrothecium verrucaria, respectively, shows higher turnover activity towards bilirubin compared to other bacterial MCOs. The current density for O2 reduction, when immobilized in a redox hydrogel, is only 12% smaller than the current obtained with Trachyderma tsunodae BOD. Under continuous electrocatalysis, an electrode modified with the new BOD is more stable, and has a higher tolerance towards NaCl, than a T. tsunodae BOD modified electrode. This makes BOD from B. pumilus an attractive new candidate for application in biofuel cells (BFCs) and biosensors.

► We identified a new bacterial bilirubin oxidase from Bacillus pumilus.
► We present a full characterization of this enzyme in solution and on modified electrodes.
► The current for O2 reduction is 68% higher than for Myrothecium verrucaria.
► The modified electrode is 400% more stable under continuous operation than other BOD.