Stabilisation of pyranose oxidase from T. multicolor
Abstract
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.
keywords Cellaldose-Oxidoreduktasen Sclerotium
Publikationen
Immobilization of catalase on different solid supports
Autoren: Gergana Delcheva Jahr: 2005
Master / Diploma Thesis
Measurement of the reduction potential of pyranose oxidase from the white rot fungus Trametes multicolor
Autoren: Prongjit, M., Sucharitakul, J., Haltrich, D., Chaiyen, P. Jahr: 2005
Chapter in collected volumes
Different strategies for the recombinant production of the tetrameric enzyme pyranose oxidase
Autoren: Ertl, S., Haltrich, D., Ludwig, R. Jahr: 2005
Conference & Workshop proceedings, paper, abstract
Pyranoses oxidase from the white-rot fungus Trametes multicolor - cloning, expression, structure, biocatalysis.
Autoren: Leitner, C., Kujawa, M., Halada, P., Volc, J., Hallberg, M., Divne, C., Haltrich, D., Peterbauer, C.K. Jahr: 2005
Conference & Workshop proceedings, paper, abstract
Pyranose oxidase from Trametes multicolor - cloning, expression, structure, biocatalysis.
Autoren: Leitner, C., Kujawa, M., Henikl, S., Halada, P., Volc, J., Hallberg M., Divne, C., Haltrich, D., Kulbe, K.D., Peterbauer, C.K. Jahr: 2005
Conference & Workshop proceedings, paper, abstract
Pyranose oxidase from Trametes multicolor - application in biocatalysis.
Autoren: M. Kujawa, C. Leitner, P. Halada, J.Volc, B.M. Hallberg, C. Divne, C.K. Peterbauer, D. Haltrich Jahr: 2005
Conference & Workshop proceedings, paper, abstract
Immobilisation of pyranose oxidase on solid supports.
Autoren: Sukyai, P., Haltrich, D., Ludwig, R. Jahr: 2005
Conference & Workshop proceedings, paper, abstract
Increased space-time yields and operational stability in flavoenzyme dehydrogenase/oxidase reactions by a laccase-mediator system
Autoren: Ludwig, R., Kulbe, K. D., Haltrich, D. Jahr: 2006
Conference & Workshop proceedings, paper, abstract
New biotransformations of some reducing sugars to the corresponding (di)dehydro(glycosyl) aldoses or aldonic acids using fungal pyranose dehydrogenase
Autoren: Sedmera, P., Halada, P., Kubátová, E., Haltrich, D., Prikrylová, V., Volc, J. Jahr: 2006
Journal articles
Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthoderma.
Autoren: Kujawa, M; Volc, J; Halada, P; Sedmera, P; Divne, C; Sygmund, C; Leitner, C; Peterbauer, C; Haltrich, D; Jahr: 2007
Journal articles
Project staff
Dietmar Haltrich
Univ.Prof. Dipl.-Ing. Dr.techn. Dietmar Haltrich
dietmar.haltrich@boku.ac.at
Tel: +43 1 47654-75211
Project Leader
01.07.2002 - 31.10.2006
BOKU partners
External partners
Institute of Microbiology, Academy of Sciences of the Czech Republic
none
partner
Department of Biotechnology
Dr. Christina DIVNE
partner