Enhanced expression of a recombinant multicopper oxidase, CueO, from Escherichia coli and its laccase activity towards aromatic substrates

Abstract

CueO from Escherichia coli belongs to multicopper oxidase family, which has gained considerable attention in the field of biotechnology. In earlier studies of recombinant CueO, the enzyme exhibits a relatively broad substrate specificity, but low activity. In this study, we establish an efficient and reliable expression system of highly active recombinant CueO by use of CueO-gene-disrupted E. coli in combination with microaerobic culture. Microaerobic culture of the recombinant E. coli generates recombinant CueO (4Cu-CueO) with four Cu ions incorporated in its T1, T2, and T3 sites. This recombinant CueO has a higher oxidation activity than a previously reported CueO. Kinetic analysis demonstrates that the high oxidation activity of 4Cu-CueO is attributable to the increase in its turnover number. Substrate screening reveals that 4Cu-CueO accepts a wide variety of compounds as substrates compared to the earlier reported CueO. The present findings suggest that the CueO expressed under the microaerobic conditions gets to be able to maximize its oxidation activity, resulting in a possible versatile biocatalyst for the oxidative polymerization, polymer functionalization and so on.