Analysis of Agaricus meleagris pyranose dehydrogenase N-glycosylation sites and performance of partially non-glycosylated enzymes

Publication date: Available online 22 January 2017 Source:Enzyme and Microbial Technology Author(s): Christoph Gonaus, Daniel Maresch, Katharina Schropp, Peter ÓConghaile, Dónal Leech, Lo Gorton, Clemens K. Peterbauer Pyranose Dehydrogenase 1 from the basidiomycete Agaricus meleagris (AmPDH1) is an oxidoreductase capable of oxidizing a broad variety of sugars. Due to this and its ability of dioxidation of substrates and no side production of hydrogen peroxide, it is studied for use in enzymatic bio-fuel cells. In-vitro deglycosylated AmPDH1 as well as knock-out mutants of the N-glycosylation sites N75 and N175, near the active site entrance, were previously shown to improve achievable current densities of graphite electrodes modified with AmPDH1 and an osmium redox polymer acting as a redox mediator, up to 10-fold. For a better understanding of the role of N-glycosylation of AmPDH1, a systematic set of N-glycosylation site mutants was investigated in this work, regarding expression efficiency, enzyme activity and stability. Furthermore, the site specific extend of N-glycosylation was compared between native and recombinant wild type AmPDH1. Knocking out the site N252 prevented the attachment of significantly extended N-glycan structures as detected on polyacrylamide gel electrophoresis, but did not significantly alter enzyme performance on modified electrodes. This suggests that not the molecule size but other factors like accessibility of the active site improved ...

Source: Enzyme and Microbial Technology - January 21, 2017 Category: Biotechnology Source Type: research