Pyranose dehydrogenase for bio-fuelcells
Abstract
Evolution and engineering of pyranose dehydrogenase for biofuel cell applications Enzymatic biological fuel cells (biofuel cells) are electrochemical devices that transform chemical to electrical energy via enzymatic catalysis. Microscale enzymatic biofuel cells are a promising future power source for implantable medical devices such as the cardiac pacemaker or prosthetic applications (artificial hearing or vision), as well as for power generation from ambient fuels for small electronic devices. In enzymatic biofuel cells, suitable redox enzymes are connected to electrodes by redox active substances (mediators) such as osmium redox polymers. Glucose oxidase is most commonly used as a model enzyme. There are, however, a number of alternative carbohydrate oxidoreductases that offer distinct advantages over glucose oxidase. One of them is pyranose dehydrogenase (PDH), a flavin-dependent oxidoreductase from litter-decomposing Agaricaceae. PDH is unable to utilize oxygen as electron acceptor and is capable of oxidizing a broad variety of monosaccharides, oligosaccharides and glycosides. Additionally, PDH oxidizes glucose and other sugars at more than one C-atom, increasing the attainable electron output per molecule of substrate. The suitability of PDH for biofuel cell applications has already been demonstrated, resulting in a biofuel cell with increased coulombic efficiency. We propose to engineer and improve certain properties of PDH for use in biofuel cells employing random strategies as well as semi-rational protein engineering. A mutant library will be constructed by a combination of error-prone PCR and gene shuffling (recombination), and screened in microtiterplate-format using a standard assay with suitable modifications for improved catalytic activity, improved stability of the biocatalyst, improved activity at lower temperatures and improved activity with primary oxidation products of PDH and other enzymes such as gluconic acid, 2-keto-glucose and 2-keto-gluconic acid. Selected variants with improved properties will be characterized with respect to several properties, especially kinetic constants for various electron acceptors such as cycloruthenate or complexed Os3+. We will also employ semi-rational protein engineering in order to improve certain properties of PDH. As soon as a three-dimensional structure of PDH is available, amino acid residues at the active site of PDH will be selected and subject to saturation mutagenesis and screening of the resulting mutant libraries for improvement of the above mentioned properties. Additionally we will target amino acid residues that display a high flexibility upon thermal motion by saturation mutagenesis, and screen for increased thermostability as a result of amino acids conferring increased rigidity. Variants of interest will also be characterized electrochemically using redox polymers with different redox potentials, and prototype biofuel cells consisting of a PDH-based anode and a suitable cathode will be constructed and their performance tested.
keywords pyranose dehydrogenase bio-fuelcells enzyme evolution enzyme engineering
Publikationen
Agaricus bisporus pyranose dehydrogenase - recombinant expression in Pichia pastoris and characterization
Autoren: Gonaus, C., Peterbauer, C.K., Haltrich, D. Jahr: 2012
Conference & Workshop proceedings, paper, abstract
Project staff
Clemens Karl Peterbauer
Assoc. Prof. Dr. Clemens Karl Peterbauer
clemens.peterbauer@boku.ac.at
Tel: +43 1 47654-75212
BOKU Project Leader
01.05.2011 - 30.04.2014
BOKU partners
External partners
Royal Institute of Technology, Department of Biochemistry and Biotechnology
Christina Divne
partner
University Lund, Department of Biotechnology, Centre for Chemistry and Chemical Engineering
Lo Gorton
partner