Stabilisation of pyranose oxidase from T. multicolor

The fungal flavoenzyme pyranose oxidase (P2O), which oxidises various sugars at position C-2 while electrons are transferred to oxygen yielding hydrogen peroxide, is of biotechnological interest for applications in food technology, e.g., for the production of beneficial probiotic food ingredients such as tagatose which can positively affect health and well-being of the consumer. Furthermore P2O can be used for certain pharmaceutical applications, such as for the formation of compounds that are thought to be efficient targets for chemotherapy of malaria. One of the drawbacks of this biocatalytically attractive enzyme, however, is its stability under operational conditions. Preliminary studies have shown that one possible reason for this inactivation is chemical modification of the biocatalyst during substrate turnover. It is the objective of this project to stabilise P2O from the white-rot fungus Trametes multicolor under operational conditions by two different approaches. The first approach is aiming at identifying amino acid residue(s) that are modified during the catalytic action of the enzyme, e.g., due to oxidation by the reactive reaction product hydrogen peroxide, by using mass spectrometry and to subsequently alter these susceptible amino acid residue(s) by site-directed mutagenesis. The recombinant and altered enzymes will be characterised pertaining to their catalytic properties and will be compared to the wild-type enzyme, especially with regard to stability under operational conditions. In the second approach an alternative electron acceptor and not oxygen will be used for substrate turnover by P2O, thus the formation of hydrogen peroxide will be avoided. Alternative electron acceptors considered will include variously substituted benzoquinones, some of which are better substrates of P2O than the presumed natural substrate oxygen, and complexed metal ions such as ferricenium. To make possible the use of these electron acceptors in low catalytic amounts, innovative reaction engineering based on a second regenerative enzyme for the continuous regeneration of the electron acceptor is necessary, which will be developed in this project. In addition to these two approaches of stabilising P2O it is planned to determine the crystal structure of the enzyme in a collaboration with a Swedish partner.

Pyranose oxidase (P2O) is an enzyme that is taking part in lignocellulose (wood) degradation of many higher fungi. P2O has found a number of interesting applications, for example it is used in clinical analytics for the determination of a marker for diabetes, or in industrial biotechnology where it serves as a biocatalyst for specific sugar oxidations. Some of these oxidised sugar compounds are attractive intermediates for further transformations. As an example one can give the reaction with galactose, which is found as a component in the milk sugar lactose. Galactose can be specifically oxidised by pyranose oxidase to 2-keto galactose, which in turn can be transformed into tagatose. Tagatose is of great interest for food applications. It is a naturally occurring sugar (its found in some berries in low concentration), it is equally sweet as table sugar (sucrose) but contains less calories, is not cariogenic (does not cause dental decay), and has proven prebiotic properties, which means that it can positively stimulate beneficial intestinal bacteria such as bifidobacteria. Pyranose oxidase is, however, not very stable in technical processes, as one of the reaction products, hydrogen peroxide, is a very reactive molecule and inactivates P2O rapidly. The aim of this project was to study this inactivation mechanism, and to stabilise the enzyme so that it is better adjusted to technical processes. We could identify an amino acid that is oxidised by hydrogen peroxide and seems to play a central role in inactivation, however this amino acid seems to play an important role in the reaction mechanism of the enzyme and cannot be replaced by another amino acid. Further studies dealt with an alternative reaction technology that avoids the formation of hydrogen peroxide, thereby stability in technical processes could be increased by a factor of five. Here alternative electron acceptors are used instead of oxygen. In the frame of this project we also studied a novel enzyme, pyranose dehydrogenase, in some detail. This enzyme is characterised by an even higher substrate tolerance than pyranose oxidase, and it could be of interest for the production of lactulose, a prebiotic compound that is also used for pharmaceutical applications. Both pyranose oxidase and pyranose dehydrogenase show promise as anodic component of biofuel cells. We could show in this project that both enzymes can communicate with electrodes, i.e., electrons are transferred from the enzyme to the electrode through redox active polymers. These biofuel cells could serve as independent small energy devices (batteries), for example in medical applications.