A Study on the Lateral Channel of Scytalidium Catalase

Abstract

Catalases are antioxidant enzymes. Their main function is to convert hydrogen peroxide, one of the reactive oxygen species, into water and oxygen. A bifunctional catalase enzyme from Scytalidium thermophilum, which belongs to the monofunctional catalase family, exhibits oxidase activity in the absence of hydrogen peroxide without the need for a cofactor. It is able to oxidize a variety of ortho- and para-diphenolic compounds, but the oxidase activity is very low (~ 10,000-fold) compared to its main activity in peroxide degradation. In this study, an attempt was made to improve the oxidase activity of catalase for its industrial utilization. For this purpose, the residues located in the lateral channel (H214, D224, and K248), which are thought to play an important role in the access of oxidase substances to the active site, were selected and converted into an amino acid with a smaller side chain -Ala- by site-directed mutagenesis. The changes in the catalytic efficiency of the variants were evaluated by spectrophotometric analysis. The results showed that oxidase activity was increased two- to threefold in all variants, while some exhibited decreased catalase activity. The D224A variant gave the best results as it showed approximately 2-fold increase in oxidase activity without loss of catalase activity (about 92% of the wild-type catalase activity was retained). Thus, a protein variant with improved properties was successfully produced.