title: Recombinantly produced cellobiose dehydrogenase from Corynascus thermophilus for glucose biosensors and biofuel cells.
abstract: Cellobiose dehydrogenase (CDH) is an emerging enzyme in the field of bioelectrocatalysis. Due to its flexible cytochrome domain, which acts as a built-in redox mediator, CDH is capable of direct electron transfer (DET) to electrode surfaces. This rare property is employed in mediatorless "third generation" biosensors. The ability of Corynascus thermophilus CDH to oxidize glucose under physiological conditions makes it a promising candidate for miniaturized glucose biosensors or glucose powered biofuel cell anodes. We report for the first time the electrochemical application and characterization of a recombinantly produced CDH in a glucose biosensor. Recombinant CDH from C. thermophilus (rCtCDH) was expressed by the methylotrophic yeast Pichia pastoris (376 U L(-1) , 132 mg L(-1) ). A comparative characterization of rCtCDH and CtCDH shows identical pH optima, K(M) values and heme b midpoint potentials. In contrast, the specific activity of rCtCDH (2.84 U mg(-1) ) and consequently the turnover numbers were ~five-times lower than for CtCDH, which was caused by a sub-stoichiometric occupation of catalytic sites with flavin-adenin-dinukleotid (FAD). The performance of rCtCDH-modified electrodes demonstrates the suitability for electrochemical studies. This opens the possibility to engineer the substrate specificity of C. thermophilus CDH for specific carbohydrates by rational engineering or directed evolution.
authors: Food Biotechnology Laboratory, Department of Food Sciences and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.
authors: Wolfgang
authors: W
authors: Harreither
authors: Alfons K G
authors: AK
authors: Felice
authors: Regina
authors: R
authors: Paukner
authors: Lo
authors: L
authors: Gorton
authors: Roland
authors: R
authors: Ludwig
authors: Christoph
authors: C
authors: Sygmund
mesh_terms: D001674:Bioelectric Energy Sources; D015374:Biosensing Techniques / Q000379:methods*; D002237:Carbohydrate Dehydrogenases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002475:Cellobiose / Q000378:metabolism; D004566:Electrodes; D005285:Fermentation; D005947:Glucose / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D020037:Sordariales / Q000201:enzymology* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D002475:Cellobiose; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase; D005947:Glucose