Electrochemical Immobilisation of Glucose Oxidase for the Controlled Production of H₂O₂ in a Biocatalytic Flow Reactor

Abstract

Electrochemical methods can be used to selectively modify the surfaces of electrodes, enabling the immobilisation of enzymes on defined areas on the surfaces of electrodes. Such selective immobilisation methods can be used to pattern catalysts on surfaces in a controlled manner. Using this approach, the selective patterning of the enzyme glucose oxidase on the electrodes was used to develop a flow reactor for the controlled delivery of the oxidant H2O2. GOx was immobilised on a glassy carbon electrode using polypyrrole, silica films, and diazonium linkers. The rate of production of H2O2 and the stability of the response was dependent on the immobilisation method. GOx encapsulated in polypyrrole was selected as the optimal method of immobilisation, with a rate of production of 91±11 μM h−1 for 4 hours of continuous operation. The enzyme was subsequently immobilised on carbon rod electrodes (surface area of 5.76 cm2) using a polypyrrole/Nafion® film and incorporated into a flow reactor. The rate of production of H2O2 was 602±57 μM h−1, with 100 % retention of activity after 7 h of continuous operation, demonstrating that such a system can be used to prepare H2O2 at continuous and stable rate for use in downstream oxidation reactions.

Coupled immobilized bi-enzymatic flow reactor employing cofactor regeneration of NAD+ using a thermophilic aldehyde dehydrogenase and lactate dehydrogenase

“…The immobilized GOx lost 27% of its initial activity after 6 hours of continuous operation in a flow system, 35 indicating that LDH immobilized in PEDOP was more stable. On examining the activity of the PEDOP-LDH-GRE in batch mode, the specific activity of immobilized LDH in PEDOP was 0.026 ± 0.004 U mg −1 , compared with a value of 3.6 ± 0.3 U mg −1 for LDH@agarose, indicating that diffusional limitation of the cofactor in the polymer layer significantly reduced the activity of PEDOP-LDH-GRE.…”

“…A flow reactor was designed to incorporate the GRE as described previously. 35 Briefly, the reactor was constructed using acrylate-based material and consists of two caps, a cylinder (inner radius: 5 mm), and a graphite rod with an accessible surface area of 5.08 cm 2 . An Instech P720 peristaltic pump was used to pump the solution through the channel at a flow rate of 0.37 or 0.56 ml min −1 .…”

“…Previously we have shown that electrochemical methods can be used for the immobilization of enzymes in biocatalysis. 34,35 LDH was immobilized on GREs in a single step using PEDOP to produce PEDOP-LDH-GRE. The activity of PEDOP-LDH-GRE was tested in a batch system prior to flow reactor operation (Fig.…”

“…For example, lipase was immobilized electrochemically in silica films on nanoporous gold (NPG) supports and incorporated into a flow reactor for the hydrolysis of 4-nitrophenyl butyrate. 34 Recently, we demonstrated that glucose oxidase (GOx) can be immobilized in PPy films on graphite rods for the generation of hydrogen peroxide at a rate of 42 μM h −1 35 in a flow reactor.…”

Application of Immobilized Enzymes in Flow Biocatalysis for Efficient Synthesis

“…Magner's group used electrochemical methods to immobilize glucose oxidase (GOx) on the electrodes and then developed a flow reactor for controllable delivery of the oxidant H 2 O 2 (Figure 3B). 40 Immobilization of the enzyme at the commercially available screen-printed carbon electrodes (SPE) was achieved by encapsulating GOx in a polypyrrole/ Nafion film. The immobilized enzymes, PPy-GOx-NF/SPE, were incorporated into a flow reactor to catalyze the oxidation of glucose for producing H 2 O 2 .…”