Solid Binding Peptide-Guided Enzyme Immobilization for Enzymatic Cascade-based Direct Bioelectrocatalytic System

Abstract

In this study, enzyme cascade-based direct bioelectrocatalytic system has been constructed using genetic fusion methodology of solid binding peptide (SBP) tag and purposed to prove the feasibility of SBP linker to control 1) cofactor-surface orientation and 2) inter-enzyme relative orientation, as well as 3) position-selective adsorption on electrode. In the first part of the study, the interfacial DET of gold binding peptide (GBP)-fused oxidoreductase on electrode was focused and evaluated depending on fused GBP types that were varied in the respect of amino acid sequence and length, though possessing identical gold binding specificity. The four types of GBP have been fused to DET-capable enzyme, GDHγα, producing 4 different fusion GDHγα. The significant alternation in oxidative current from fusion GDHγα on electrode was observed according to introduced GBP type. Through concomitant analysis with predictive docking model, it was found that cofactor-surface interface could be obstructed or open even by slight change in surface conformation of fusion enzymes which has induced by gold-binding conformation of fused GBPs. Then, INV as the upstream enzyme of GDHγα, was incorporated at previously established DET-based fusion GDHγα electrode, for investigate inter-enzyme orientation dependent cascade reaction in direct bioelectrocatalytic system. With altering inter-enzyme relative orientation by fusion of GBP at various ends of enzymatic structure of cascadic bienzyme, intermediate delivery between active sites of coupling enzymes was validated. As two different enzymes are co-immobilized, it was observed that not only intermediate delivery route could be variable depending on relative orientation of coupling enzymes, but also different type of co-bound enzyme could exhibit unfavorable steric effect that obstruct cofactor-surface interface, interfering interfacial DET. Thus, necessity of exquisite design of bienzymatic electrode was emphasized for construction of efficient enzyme cascade-induced direct bioelectrocatalytic system. Furthermore, the viability of SBP-fusion technology for enabling regioselective enzyme binding on predetermined surface has been demonstrated, which is a critical feature for further elaborate inter-enzyme distance modulation and binding distribution in enzyme electrodes. To optimize the selective gold binding characteristics of the fusion enzyme, the GBP tandem repeats were tuned considering genetic modification-dependent biocatalytic and gold-binding activity changes. Along with qualitative evidence for the fusion enzyme's selective binding properties toward gold versus the SiO2 surface, position-specific binding of GBP-fused enzyme on micro/nano-patterned Au/SiO2 surfaces was demonstrated, indicating that SBP fusion technology is promising for highly selective surface-templated assembly of enzymes. Taken together, this work showed the efficacy of SBP fusion technology as a generic tool for developing cascade-induced direct bioelectrocatalytic system. The genetic fusion of GBP to the enzymatic structure enables surface-oriented immobilization of enzymes as well as inter-enzyme orientation when cascadic multienzymes are incorporated on single electrode. Additionally, precise arrangement of the enzyme on the electrode could be accomplished as a micro- and nanometer-scale predetermined surface is introduced as carrier.