Cloning, characterization and directed evolution of pyranose oxidase from Trametes versicolor for biofuel cell applications

  • Biofuel cells convert chemical energy to electric energy using an enzyme or a whole cell. Glucose oxidase (GOx) has become the prime enzyme used in the anodic compartment of biofuel cells. Miniaturized implanted biofuel cells have been reported losing 6 % of its power per day due to blood conditions which are suboptimal for GOxs. Prolonging the life span for such a battery is a main goal in developing these devices. This could be addressed by tailoring GOx or, alternatively, by using pyranose oxidase (POx): POxs show a broader substrate profile, a higher affinity towards glucose and no anomeric preference. POxs optimum pH is around 7 and at values superior to 7, kinetics is barely affected by buffer composition. However, the catalytic efficiency needs to be improved. Moreover, POxs are specially challenging since in contrast to GOxs there is little knowledge about POx structure, their structure-function relationships, electrochemical properties, and enzymatic stabilities. In this work, POx from Trametes versicolor was cloned from a cDNA library and was expressed in Escherichia coli as an intracellular active recombinant enzyme. Directed evolution via error-prone PCR random mutagenesis was used to generate mutant libraries, which were tested for improved catalytic efficiency via a high-throughput ABTS assay. Further cloning in a mutator E. coli strain led to a C-terminally truncated POx variant (trPOx), that exhibited a ̃14-fold increased kcat and a ̃78-fold improved catalytic efficiency. trPOx is the smallest active POx yet described. Results of this study proved the directed evolution strategy to be successful at optimizing catalytic activities of POxs. Simultaneously, a novel HTS assay based in the reaction of phenylhydrazine with 2-keto-glucose, the enzymatic product of POxs over D-glucose, was developed. This assay will allow assessing catalytic activity of O2-independent POxs. The ferrocenemethanol assay could assist tailoring POxs for O2-independency.

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Publishing Institution:IRC-Library, Information Resource Center der Jacobs University Bremen
Granting Institution:Jacobs Univ.
Author:Josefa Caballero Hernández
Referee:Matthias Ullrich, Alexander Lerchl, Ana Linares Gil
Advisor:Matthias Ullrich
Persistent Identifier (URN):urn:nbn:de:101:1-2013052812362
Document Type:PhD Thesis
Date of Successful Oral Defense:2011/01/12
Date of First Publication:2011/03/04
PhD Degree:Biochemical Engineering
School:SES School of Engineering and Science
Other Countries Involved:Spain
Library of Congress Classification:T Technology / TP Chemical technology / TP248.13-248.65 Biotechnology / TP248.3 Biochemical engineering. Bioprocess engineering

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