Sustainable production of formic acid from CO2 by a novel immobilized mutant formate dehydrogenase
Ahmet Tülek, Elif Günay, Burak Servili, Şebnem Eşsiz, Barış Bi̇nay, Deniz Yıldırım
Abstract
Formate dehydrogenase (NAD + -dependent FDH) is an enzyme that catalyzes the reversible oxidation of formate to CO 2 while reducing NAD + to NADH. The enzyme has been used in industrial and chemical applications for NADH regeneration for a long time. However, discovering the unique ability of FDHs, which is to reduce CO 2 and produce formic acid, leads studies focusing on discovering or redesigning FDHs. Despite using various protein engineering techniques, these studies mostly target the same catalytic site amino acids of FDHs. Here, for the first time, the effect of an Asp188 mutation on a potential allosteric site in NAD + -dependent Ct FDH around its subunit-subunit interface was studied by molecular modelling and simulation in the presence of bicarbonate and formate. Biochemical and kinetic characterization of this Asp188Arg mutant and wild type Ct FDH enzymes were performed in detail. Both enzymes were also immobilized on newly synthesized MWCNT-Ni-O-Si/Ald and MWCNT-Ni-O-Si/Glu supports designed to overcome well-known Ct FDH stability problems including thermostability and reuse resistance. Integrating mutation and immobilization provided about a 25-fold increase in catalytic efficiency for carbonate activity. The one-way ANOVA analysis also ensured significant effect of the mutation and immobilization on kinetic constants. After characterizing the immobilization of highly purified wild type and mutant enzyme with instrumental analysis techniques, the thermal stability of MWCNT-Ni-Si@wt- Ct FDH and MWCNT-Ni-Si@mt- Ct FDH was found to increase about 11- and 18-fold, respectively, compared to their free counterparts at 50 °C. The mutant Ct FDH and its immobilized counterpart produced around 2-fold more formic acid than those of wild type Ct FDH and its immobilized counterpart under the same conditions. MWCNT-Ni-Si@wt- Ct FDH and MWCNT-Ni-Si@mt- Ct FDH remained around 82 % and 86 % of their initial activities respectively after lots of recycling. Integration of subunit interface amino acid position of NAD + dependent FDHs engineering and immobilization provides a new insight can be scientifically and rationally employed for this current application FDHs as a solution to produce formic acids from renewable sources.