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Hyeryeong LEE,Stacy Simai REGINALD,Taehoon YI,,In Seop CHANG 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10
In this study, using the FAD-dependent glucose dehydrogenase (FAD-GDH) as a model enzyme, it is aimed to control the enzymatic orientation of the enzyme via site-specific fusion of gold binding peptide in enzyme. Most importantly, positioning of synthetized fusion protein would be regulated at nano-level, using nano-patterned electrode fabricated through e-beam lithography technique. For this, 1) the synthetic glucose dehydrogenase (GDH), consisting of the site-specific expression of a gold binding peptide (GBP) on the α-subunit of GDH which enables close proximity between enzymatic active and electrode surface as well as DET of enzyme-electrode interface will be used; 2) the number of GBP repeats fused to FAD-GDH would be optimized in terms of enzymatic catalytic activity and binding affinity; 3) The nano-patterned electrode would be fabricated via e-beam lithography for its unit pattern to have diameter similar to that of target enzyme; 3) the fusion protein will be immobilized on the nano-patterned electrode surface and the binding morphology of enzymatic nanopatterns would be investigated.
Min Ji KIM,Stacy Simai REGINALD,Hyeryeong LEE,Serah CHOI,Basit SHARIF,In Seop CHANG 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10
Over the last century, the accumulation of carbon dioxide (CO₂) which accounts for the largest portion of greenhouse gases in the atmosphere has provoked extreme global warming and environmental issues. To tackle these issues, carbon capture, utilization and storage (CCUS) technology has received considerable attention through all the related areas. In the field of CO₂ conversion having an effect on CO₂ valorization to valuable chemicals as well as removal, biocatalysts such as enzyme are promising due to their high specificity to substrate and ability of catalysis at mild conditions. Herein, we focused on CO₂ reducing enzyme, formate dehydrogenase (EC 1.17.1.9) (FDH) which can reversibly catalyze CO₂ reduction and formate oxidation. FDHs existed in a diverse array of organisms can be divided into NAD⁺-dependent and metal-dependent FDH. While metal-dependent FDH is highly efficient for CO₂ reduction to formate but requires strict anaerobic conditions, NAD⁺-dependent FDH can be easily handled, but NADH are required as a natural cofactor. In this study, we attempted to investigate the availability of Candida methylica FDH(cmFDH) for electroenzymatic CO₂ reduction to formate. Solid-binding peptide (SBP) was introduced at either N- terminus (gbp(N)-FDH) or C- terminus (FDH-gbp(C)) via protein engineering to develop a stable enzyme immobilization at the enzyme-electrode interface. The recombinant FDH and the two synthetic FDHs are characterized for their biocatalytic activity. The results indicated that GBP fusion CmFDHs retain or improve their enzyme activities. Cyclic voltammetry (CV) result shows that the electrons can be directly transferred from FDHs to the electrode surface and vice versa for formate oxidation and CO₂ reduction in the absence of NADH.
Lee, Yoo Seok,Baek, Seungwoo,Lee, Hyeryeong,Reginald, Stacy Simai,Kim, Yeongeun,Kang, Hyunsoo,Choi, In-Geol,Chang, In Seop American Chemical Society 2018 ACS APPLIED MATERIALS & INTERFACES Vol.10 No.34
<P>Direct electron transfer (DET) between enzymes and electrodes is a key issue for practical use of bioelectrocatalytic devices as a bioenergy process, such as enzymatic electrosynthesis, biosensors, and enzyme biofuel cells. To date, based on the DET of bioelectrocatalysis, less than 1% of the calculated theoretical current was transferred to final electron acceptor due to energy loss at enzyme-electrode interface. This study describes the design and construction of a synthetic glucose dehydrogenase (GDH; α and γ subunits) combined with a gold-binding peptide at its amino or carboxy terminus for direct contact between enzyme and electrode. The fused gold-binding peptide facilitated stable immobilization of GDH and constructed uniform monolayer of GDH onto a Au electrode. Depending on the fused site of binding peptide to the enzyme complex, nine combinations of recombinant GDH proteins on the electrode show significantly different direct electron-transfer efficiency across the enzyme-electrode interface. The fusion of site-specific binding peptide to the catalytic subunit (α subunit, carboxy terminus) of the enzyme complex enabled apparent direct electron transfer (DET) across the enzyme-electrode interface even in the absence of the electron-transfer subunit (i.e., β subunit having cytochrome domain). The catalytic glucose oxidation current at an onset potential of ca. (−)0.46 V vs Ag/AgCl was associated with the appearance of an flavin adenine dinucleotide (FAD)/FADH<SUB>2</SUB> redox wave and a stabilized bioelectrocatalytic current of more than 100 μA, determined from chronoamperometric analysis. Electron recovery was 7.64%, and the catalytic current generation was 249 μA per GDH enzyme loading unit (U), several orders of magnitude higher than the values reported previously. These observations corroborated that the last electron donor facing to electrode was controlled to be in close proximity without electron-transfer intermediates and the native affinity for glucose was preserved. The design and construction of the site-specific “sticky-ended” proteins without loss of catalytic activity could be applied to other redox enzymes having a buried active site.</P> [FIG OMISSION]</BR>