Construction of Uniform Monolayer- and Orientation-Tunable Enzyme Electrode by a Synthetic Glucose Dehydrogenase without Electron-Transfer Subunit via Optimized Site-Specific Gold-Binding Peptide Capable of Direct Electron Transfer

Yoo Seok Lee, Seungwoo Baek, Hyeryeong Lee, Stacy Simai Reginald, Yeongeun Kim, Hyunsoo Kang, In-Geol Choi, In Seop Chang

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

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)/FADH2 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.

Original languageEnglish
Pages (from-to)28615-28626
Number of pages12
JournalACS Applied Materials and Interfaces
Volume10
Issue number34
DOIs
Publication statusPublished - 2018 Aug 29

Fingerprint

Glucose 1-Dehydrogenase
Enzyme electrodes
Gold
Peptides
Glucose
Monolayers
Enzymes
Electrons
Electrodes
Biological fuel cells
Proteins
Oxidoreductases
Recombinant proteins
Flavin-Adenine Dinucleotide
Cytochromes
Biosensors
Catalyst activity
Energy dissipation
Fusion reactions
Binding Sites

Keywords

  • direct electron transfer
  • electron tunneling distance
  • gold-binding peptide
  • orientation
  • synthetic glucose dehydrogenase

ASJC Scopus subject areas

  • Materials Science(all)

Cite this

Construction of Uniform Monolayer- and Orientation-Tunable Enzyme Electrode by a Synthetic Glucose Dehydrogenase without Electron-Transfer Subunit via Optimized Site-Specific Gold-Binding Peptide Capable of Direct Electron Transfer. / Lee, Yoo Seok; Baek, Seungwoo; Lee, Hyeryeong; Reginald, Stacy Simai; Kim, Yeongeun; Kang, Hyunsoo; Choi, In-Geol; Chang, In Seop.

In: ACS Applied Materials and Interfaces, Vol. 10, No. 34, 29.08.2018, p. 28615-28626.

Research output: Contribution to journalArticle

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abstract = "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)/FADH2 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.",
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