Recent Advances in Oxidoreductases Biochemistry and Biotechnology

The multifunctional catalytic hemoglobin from the terebellid polychaete Amphitrite ornata, also named dehaloperoxidase (AoDHP), utilizes the typical oxygen transport function in addition to four observed activities involved in substrate oxidation. The multifunctional ability of AoDHP is presently a rare observation, and there exists a limitation for how novel dehaloperoxidases can be identified from macrobenthic infauna. In order to discover more infaunal DHP-bearing candidates, we have devised a facilitated method for an accurate taxonomic identification that places visual and molecular taxonomic approaches in parallel. Traditional visual taxonomic species identification by the non-specialist, at least for A. ornata or even for other marine worms, is a very difficult and time-consuming task since a large diversity is present and the method is restricted to adult worm specimens. The work herein aimed to describe a method that simplifies the taxonomic identification of A. ornata in particular through the assessment of its mitochondrial cytochrome c oxidase subunit I gene by employing the DNA barcoding technique. Furthermore, whole-worm specimens of A. ornata were used to extract and purify AoDHP followed by an H₂O₂-dependent peroxidase activity assay evaluation against substrate 2,4,6-trichlorophenol. AoDHP isoenzyme A was also overexpressed as the recombinant protein in Escherichia coli, and its peroxidase activity parameters were compared to AoDHP from the natural source. The activity assay assessment indicated a tight correlation for all Michaelis–Menten parameters evaluated. We conclude that the method described herein exhibits a streamlined approach to identify the polychaete A. ornata, which can be adopted by the non-specialist, and the full procedure is predicted to facilitate the discovery of novel dehaloperoxidases from other marine invertebrates. Full article

Pyranose oxidase (POx) is an FAD-dependent oxidoreductase and belongs to the glucose–methanol–choline (GMC) superfamily of oxidoreductases. As recently reported, POxs and FAD-dependent C-glycoside oxidases (CGOxs) share the same sequence space, and phylogenetic analysis of actinobacterial sequences belonging to this shared sequence space showed that it can be divided into four clades. Here, we report the biochemical characterization of a POx/CGOx from Microbacterium sp. 3H14 (MPOx), belonging to the hitherto unexplored clade II of actinobacterial POx/CGOx. Overall, MPOx demonstrates comparable features to POxs/CGOxs of clades III and IV, including the preference for glycosides over monosaccharides as electron donors. However, as MPOx efficiently oxidizes the C-glycoside aspalathin as well as the O-glycoside phlorizin, it shows activity with yet another set of glycoside structures compared to other POx/CGOx members. Full article

Intracellular alcohol oxidase (AOX) was isolated from the basidiomycetous white rot fungus Cerrena unicolor FCL139. The enzyme was semi-purified (13-fold) using two-step chromatography with 30% activity recovery. The identity of the protein was confirmed by LC-MS/MS analysis, and its MW (72 kDa) and pI (6.18) were also determined. The kinetics parameters of the AOX reaction towards various substrates were analysed, which proved that, in addition to methanol (4.36 ± 0.27% of the oxidised substrate), AOX most potently oxidises aromatic alcohols, such as 4-hydroxybenzyl alcohol (14.0 ± 0.8%), benzyl alcohol (4.2 ± 0.3%), anisyl alcohol (7.6 ± 0.4%), and veratryl alcohol (5.0 ± 0.3%). Moreover, the influence of selected commercially available proteases on the biocatalytic properties of AOX from C. unicolor was studied. It was proved that the digested enzyme lost its catalytic potential properties except when incubated with pepsin, which significantly boosted its activity up to 123%. Full article