P2O

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About the enzyme

Pyranose oxidase (P2O, EC 1.1.3.10) is a flavin-dependent oxidoreductase of the glucose-methanol-choline (GMC) superfamily of oxidoreductases. It is responsible for oxidizing D-glucose and other monosaccharide substrates at the C2 position and reducing molecular oxygen to hydrogen peroxide. P2O is found in wood-degrading fungi. The hydrogen peroxide produced by P2O is considered to be a fuel for other enzymes that degrade lignocellulose, so it is also a member of the auxiliary active family 3 (AA3_4). The enzyme was discovered in the mycelium of Spongipellis unicolor in 1968. The crystal structure of P2O from fungi can be obtained from PDB. In addition, the structure of many P2O variants has also been resolved.

Like other members of the GMC family, P2O is composed of a flavin-binding domain and a substrate-binding domain. The flavin-binding domain contains a βαβ mononucleotide-binding motif and is highly conserved throughout the flavoproteins. The substrate binding domain is the opposite, showing more sequence variation, reflecting the preference for different electron-donor substrates. Fungal P2O protein is formed by four identical monomers, each of which contains a flavin-adenine dinucleotide (FAD) covalently linked to a histidine residue in the active site. A big central void is observed in the tetrameric structure of fungal P2O, which connects to all four active sites. This central void is the necessary channel for the substrate to enter the active site. The interactions between the individual subunits that form the homo-tetramer are carried out by oligomerisation structures that include an oligomerisation arm that extends to surround adjacent subunits, and a loop providing subunit-subunit interactions and a small domain termed head domain.

Figure 1. Two subunits of the tetrameric pyranose oxidase from Trametes ochracea (1TT0) showing the central void (black frame) that isolates the active site entrances from the exterior (Abrera, A.T.; et al. 2020)

Figure 2. Close-up of the entrance to channel leading into to interior void of pyranose oxidase from Trametes ochracea (1TT0) (Abrera, A.T.; et al. 2020)

Previous and current use in biosensors

The development of P2O-based biosensors has always been the subject of related research, including the discovery of novel enzymes, the production of variants, and the enhancement of electron transfer, etc. At present, there are more and more applications of P2O-based equipment to detect various biomolecules, and they have applications in the food industry, disease diagnosis, biological process monitoring and screening, etc. P2O, as a biological component for biosensor, has been proven on various types of electrodes including glassy carbon, platinum, gold, and screen-printed electrodes. It is still being evaluated on other platforms to improve the overall biological performance of the sensor. The reason why P2O can be used as a popular biosensor is mainly due to its reaction with glucose. However, since P2O can also detect other sugars, the application of P2O-based biosensors has also been extended to the detection of other carbohydrates.

Exclusively different P2O of fungal origin was used in these biosensor studies. In the study of Lidén et al., P2O from P. chrysosporium (PcP2O) and horseradish peroxidase (HRP) were co-immobilized on a carbon paste electrode (CPE), both H₂O₂ and glucose was detected using this bi-enzymatic biosensor. The H₂O₂ formed by the reaction of P2O and glucose is absorbed by HRP and then transfers electrons between the HRP and the graphite electrode. This b-enzymatic biosensor shows a good response to various carbohydrates. Many years later, scientists have developed P2O-based biosensors for use in food and beverages to monitor various food processing processes. P2O (CsP2O) from Coriolus sp. immobilized on a carbon paste electrode was first applied to detect glucose, galactose, mannose, xylose, and maltose. 

Greatest problems of the enzyme and the biosensor

The oxygen turnover rate of P2O is lower than that of GOx, but it shows considerable activity to oxygen. The reduction of oxygen to hydrogen peroxide in the catalytic process will interfere with its sensitivity, especially at low potentials. The reaction with oxygen is not a required characteristic for biosensor and BFC applications, as it may cause electron leakage. In addition, H2O2 may cause oxidative damage, thereby causing enzyme inactivation. In order to solve this problem, scientists successfully used enzyme engineering to modify the oxygen reactivity of P2O.