Spectroelectrochemical Investigation of GMC Flavoenzymes

• Applicant: Professor Dr. Thomas Lenzer, since 7/2020
• Subject Area: Analytical Chemistry ; Biological and Biomimetic Chemistry ; Physical Chemistry of Solids and Surfaces, Material Characterisation
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 401882662

The aim of this project is the spectroelectrochemical investigation of flavoenzymes belonging to the family of glucose, methanol, or choline (GMC) oxidizing oxidoreductases. By spectroelectrochemistry the pH-dependent redox potentials of these enzymes, respectively their individual domains can be determined. For this purpose a home-made LabView controlled spectroelectrochemical setup shall be further developed to do the measurements not only in the potentiostatic but also in the coulometric working mode. In this context also the sensitivity of the setup needs to be increased to measure the transferred charge for each titration step. To reach electrochemical equilibrium spectroelectrochemical measurements have to be carried out in the presence of redox mediators, which are small molecules that shuttle the electrons between the electrochemically active centers of the enzyme and the electrode. As, depending on the desired potential range, suitable redox mediators are not always commercially available, they need to be synthesized. These mediators shall be applied freely diffusing in solution and for comparison also tethered to the electrode surface via long and flexible linkers.As a model enzyme to establish the coulometric working mode we will use glucose oxidase (GOx), which we have investigated previously in the potentiostatic working mode. GOx from Aspergillus niger is the most frequently studied redox enzyme, and it is of particular importance for applications as catalyst in electrochemical glucose biosensors or at the anode of glucose biofuel cells. Based on the results obtained with GOx we will compare potentiostatic and coulometric working mode and figure out their individual advantages. The improved spectroelectrochemical setup will be used to investigate further analytically relevant members of the GMC family beginning with glucose oxidizing enzymes, i.e. FAD-dependent glucose dehydrogenase from Aspergillus flavus, different cellobiose dehydrogenases from ascomycetes, comprising a flavin and a heme domain, and FAD-dependent glucose dehydrogenase from Burkholderia cepacia with three individual domains.In addition, alcohol oxidase (AOx), cholesterol oxidase (ChOx), and choline oxidase (COx) will be investigated. AOx catalyzes the oxidation of short chain alcohols such as methanol, ethanol or 1-propanol and is of particular interest for biofuel cell applications. ChOx is often used for cholesterol determination and is the second most frequently used enzyme in clinical laboratories (behind GOx). COx catalyzes the oxidation of choline to betaine aldehyde as well as the consecutive oxidation from betaine aldehyde to the corresponding acid resulting in a transfer of four electrons per substrate molecule in total. The expected results will help to understand the basic electrochemical properties of these enzymes and pave the way for strategies to “wire” these enzymes to electrodes (as required for biosensor and biofuel cell applications).

• DFG Programme: Research Grants
• Ehemaliger Antragsteller: Dr. Gilbert Nöll, until 7/2020