Sequential Co-immobilization of Enzymes in Metal-Organic Frameworks for Efficient Biocatalytic Conversion of Adsorbed CO2 to Formate

Abstract

The main challenges in multienzymatic cascade reactions for CO₂ reduction are the low CO₂ solubility in water, the adjustment of substrate channeling, and the regeneration of co-factor. In this study, metal-organic frameworks (MOFs) were prepared as adsorbents for the storage of CO₂ and at the same time as solid supports for the sequential co-immobilization of multienzymes via a layer-by-layer self-assembly approach. Amine-functionalized MIL-101(Cr) was synthesized for the adsorption of CO₂. Using amine-MIL-101(Cr) as the core, two HKUST-1 layers were then fabricated for the immobilization of three enzymes chosen for the reduction of CO₂ to formate. Carbonic anhydrase was encapsulated in the inner HKUST-1 layer and hydrated the released CO₂ to ${\text{HCO}}_3^{-}$ . Bicarbonate ions then migrated directly to the outer HKUST-1 shell containing formate dehydrogenase and were converted to formate. Glutamate dehydrogenase on the outer MOF layer achieved the regeneration of co-factor. Compared with free enzymes in solution using the bubbled CO₂ as substrate, the immobilized enzymes using stored CO₂ as substrate exhibited 13.1-times higher of formate production due to the enhanced substrate concentration. The sequential immobilization of enzymes also facilitated the channeling of substrate and eventually enabled higher catalytic efficiency with a co-factor-based formate yield of 179.8%. The immobilized enzymes showed good operational stability and reusability with a cofactor cumulative formate yield of 1077.7% after 10 cycles of reusing.

Keywords: CO2 reduction; improved conversion; metal-organic framework; sequential co-immobilization of enzymes; storage of CO2.