Improving the biocatalyst. Engineering of fungal oxidoreductases and thin-film electrodes for improvement of membraneless biofuel cells and carbohydrate biosensors.

Roberto Ortiz

Research output: ThesisDoctoral Thesis (compilation)


This thesis presents investigations for the improvement of electrical contact and catalytical properties of two fungal redox enzymes, cellobiose dehydrogenase (CDH) and pyranose dehydrogenase (PDH), with electrodes and the use of the prepared electrodes as carbohydrate biosensors and in enzymatic biofuel cells. The performance of enzyme modified electrodes can be improved in two ways, by modifying the bioelement or by modifying the electrode. The thesis presents advancements and new techniques on both the enzymes used as well as the electrodes.
Electrodes were modified either with drop-cast gold nanoparticles (AuNPs), single-walled carbon nanotubes (SWCNT) or carbon nanoparticles (CNP) to increase the surface area onto which CDH can be immobilized. A novel method is presented for covalent immobilization of CDHs onto AuNPs modified gold electrodes with a mixture of thiol based self-assembled monolayers (SAMs) and cross-linked with glutaraldehyde. The standard rate constant (kS) for CDH was calculated for the first time, an enzymatic fuel cell (EFC) bioanode operating in human physiological fluids was tested and the EFC was able to power a wireless device measuring glucose. CDH was adsorbed onto glassy carbon electrodes with drop-cast SWCNT chemically grafted with aryl diazonium salts. A lower onset potential for the turn-over and a higher current density (J) were observed for grafted compared to non-grafted electrodes and the highest J in direct electron transfer (DET) mode at physiological pH for any CDH was presented. CDH was also immobilized onto CNP modified graphite electrodes to improve the lactose biosensing. The recently discovered Neurospora crassa CDH was electrochemically and kinetically characterized in DET and mediated electron transfer modes when adsorbed onto graphite electrodes and thiol based SAMs with different functionalities at different pHs. Regarding modification of enzymes two methods were investigated, deglycosylation and site-directed mutagenesis. A single mutation was introduced in the active site of CDH to improve the affinity for glucose and decrease it for maltose. The mutants were investigated adsorbed on graphite electrodes and thiol based SAMs modified gold electrodes. CDH and PDH were enzymatically deglycosylated and the effects on the electrochemical properties investigated. The elimination of the glycan shell from the enzymes showed to have a steric effect and a higher surface concentration of enzymes was possible compared to the glycosylated counterparts. Maximum J for deglycosylated CDH and PDH was greatly improved. PDH only showed DET once it was deglycosylated. Deglycosylated PDH was also investigated in MET mode using Os bound redox polymers on graphite electrodes showing higher J than reported before for glycosylated.
Original languageEnglish
Awarding Institution
  • Centre for Analysis and Synthesis
  • Gorton, Lo, Supervisor
Award date2013 Nov 29
ISBN (Print)978-91-7422-335-4
Publication statusPublished - 2013

Bibliographical note

Defence details

Date: 2013-11-29
Time: 13:00
Place: Lecture Hall B, Kemicentrum, Getingevägen 60

External reviewer(s)

Name: Bilewicz, Renata
Title: Prof. Dr.
Affiliation: Department of Chemistry, University of Warsaw, Warsaw, Poland


The information about affiliations in this record was updated in December 2015.
The record was previously connected to the following departments: Analytical Chemistry (S/LTH) (011001004), Biochemistry and Structural Biology (S) (000006142)

Subject classification (UKÄ)

  • Analytical Chemistry
  • Biological Sciences

Free keywords

  • Cellobiose dehydrogenase
  • pyranose dehydrogenase
  • biosensor
  • enzymatic biofuel cell
  • carbon nanotubes
  • gold nanoparticles
  • direct electron transfer
  • flow-injection analysis
  • diazonium salts
  • self-assembled monolayers


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