Fundamental understanding of the ways organic and/or inorganic catalysts can be incorporated with enzymes to enhance the efficiency of multi-step reactions can lead to knowledge that is crucial for any further improvements in the development of multi-component multi-step catalytic cascades. This knowledge can provide essential clues to the novel design of systems for energy conversion and harvesting as well as for production of advanced materials that require multi-step catalysis. The goal of this project is to develop strategies for spatially organizing three different catalysts (biological, molecular, and inorganic) using 3D-Graphene nanosheets (3D-GNS) as a scaffold and apply it to efficiently catalyze model catalytic cascade: multi-step oxidation of glycerol [1,2].

In this work, we first studied the immobilization of oxalate decarboxylase (OxDC) from Bacillus subtilis, through physical absorption and tethering using 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBSE) on conductive carbonaceous supports such as multi-walled carbon nanotube buckypaper and 3D-GNS. An indirect enzymatic assay using NAD-dependent formate dehydrogenase was performed to determine the kinetic parameters (K_M V_M) of immobilized OxDC onto these supports. We also employed cyclic voltammetry to study the immobilization of a molecular catalyst, pyrene-tethered TEMPO derivative (TEMPO = 2,2,6,6-tetramethylpiperidine-N-oxyl) on 3D-GNS. Furthermore, we explored optimal strategies to incorporate both OxDC and molecular catalysts in a way that will enable an efficient two-step catalytic cascade - the last two steps in the glycerol oxidation mechanism (Figure). Results of electrochemical studies show that OxDC and pyrene-TEMPO were successfully immobilized on the carbonaceous scaffold. Moreover, it was shown that this hybrid catalyst can catalyze two different reactions sequentially (i.e. conversion of oxalate to formate by OxDC/3D-GNS and oxidation of formate to CO₂ by pyrene-TEMPO/3D-GNS).

[1] S. Abdellaoui, D. P. Hickey, A. R. Stephens, and S. D. Minteer, Recombinant oxalate decarboxylase: enhancement of a hybrid catalytic cascade for the complete electro-oxidation of glycerol. Chem. Commun., 51, 14330-14333 (2015).

[2] S. Abdellaoui, M. S. Chavez, I. Matanovic, A. R. Stephens, P. Atanassov, S. D. Minteer, Hybrid molecular/enzymatic catalytic cascade for complete electro-oxidation of glycerol using a promiscuous NAD-dependent formate dehydrogenase from Candida boidinii. Chem. Commun., 53, 5368-5371 (2016).