Effect of enzymatic orientation through the use of syringaldazine molecules on multiple multi-copper oxidase enzymes

Abstract

The effect of proper enzyme orientation at the electrode surface was explored for two multi-copper oxygen reducing enzymes: Bilirubin Oxidase (BOx) and Laccase (Lac). Simultaneous utilization of "tethering" agent (1-pyrenebutanoic acid, succinimidyl ester; PBSE), for stable enzyme immobilization, and syringaldazine (Syr), for enzyme orientation, of both Lac and BOx led to a notable enhancement of the electrode performance. For Lac cathodes tested in solution it was established that PBSE-Lac and PBSE-Syr-Lac modified cathodes demonstrated approximately 6 and 9 times increase in current density, respectively, compared to physically adsorbed and randomly oriented Lac cathodes. Further testing in solution utilizing BOx showed an even higher increase in achievable current densities, thus BOx was chosen for additional testing in air-breathing mode. In subsequent air-breathing experiments the incorporation of PBSE and Syr with BOx resulted in current densities of 0.65 ± 0.1 mA cm(-2); 2.5 times higher when compared to an unmodified BOx cathode. A fully tethered/oriented BOx cathode was combined with a NAD-dependent Glucose Dehydrogenase anode for the fabrication of a complete enzymatic membraneless fuel cell. A maximum power of 1.03 ± 0.06 mW cm(-2) was recorded for the complete fuel cell. The observed significant enhancement in the performance of "oriented" cathodes was a result of proper enzyme orientation, leading to facilitated enzyme/electrode interface interactions.

Enhancement of Electrochemical Performance of Bilirubin Oxidase Modified Gas Diffusion Biocathode by Porphyrin Precursor

“…Similar strategy was extended to the BOx enzymes where the studies show that the substrate-pocket did not exhibit hydrophobic interactions but electrostatic interactions, which are an efficient way to achieve direct wiring of BOx [27]. Along this line, different literature reports have focused on incorporating specific substrates of BOx such as bilirubin [28], quinones [29], and syringaldazine [30] towards appropriate orientation that can be convenient for DET-type electrocatalysis. Our group has previously reported on crosslinking the enzyme to the electrode with orienting agents, two bilirubin functional analogues, pyrrole-2-carboxaldehyde and 2,5-dimethyl-1-phenyl-1H-pyrrole-3-carbaldehyde, for enzyme orientation and 1-pyrenebutanoic acid, succinimidyl ester (PBSE) as the tethering agent.…”

“…Thus, the electronegative N-atom from the pyrrole moiety and the Oatom from the aldehyde group can act as hydrogen bond acceptors and the H-atom as a hydrogen bond donor [29]. Subsequently, we reported on the utilization of syringaldazine (Syr), for enzyme orientation, of both Laccase and BOx that demonstrated approximately 6 and 9 times increase in current density, respectively, compared to physically adsorbed and randomly oriented Lac cathodes [30].…”

“…Glucose dehydrogenase (GDH, Toyobo) based anodes were prepared for complete fuel cell testing. Polymethylene green (PMG) was electrodeposited onto (electrode area 7.3 cm 2 ) carbon felt electrodes following a modified version of a previously described procedure [30]. A single wall nanotube (SWNT-PEI) ink solution containing GDH was drop-casted on top of the PMG treated carbon felt electrodes.…”

Untitled

“…The first approach uses modification of the electrode surface with enzyme`s natural substrate or its functional analogue, which ultimately results in proper enzyme orientation and facilitated electron transfer (Figure 2). This hypothesis was applied for the orientation of two of the most famous oxygen-reducing enzymes, Bilirubin Oxidase and Laccase and its rationality was demonstrated in the development of oxygen reducing electrodes [17,18]. Further this hypothesis was transferred towards enhancement of electron transfer from "anodic" enzymes towards solid supports, where the enzyme terminal electron acceptor was utilized for electrode surface modification.…”

Bioelectrocatalysis and More

“…A new approach for improved enzyme-electrode interactions and ultimately for enhanced bacteria-electrode electron transfer has been developed [17][18][19]. This approach is based on the specific enzyme-substrate interactions, relying on key-lock principle that is the main advantage of enzymatic catalysis.…”