Construction of Solid Binding Peptide (SBP)-Fused Non-Native Chimeric Enzyme to Promote Bienzymatic Cascade Induced Bioelectrocatalysis

Abstract

In nature, most enzymes form a multienzyme complex called “metabolon” in the cellular system. The natural metabolon has been evolved to increase the efficiency of multi-step biocatalysis involved in such conditions. Such principle of enzymatic cascade reaction could be applied to the system which mimics pertinent reaction in vitro. Especially, its pertaining to bioelectrocatalytic system is quite promising to enhance its performance in terms of electrical generation or biochemical production, depending on systematic applications. In the previous studies , it was abundantly reported that multi-enzymatic reaction has been utilized to generate higher electrical current via sequential enzymatic oxidation of substrates (e.g., methanol, glucose, etc.) in bioelectrochemical systems, and to operate biochemical production system utilizing multi-enzymic conversion or reduction reactions. However, it is still challenging to implement multi-step enzymatic reaction in the electrode platforms due to unmanageable regulations of inter-enzyme distance or electrical connection between enzymatic cofactor and electrode, during multi-enzyme co-immobilization on electrode surface.Herein, we intentionally design and construct variants of chimeric enzyme in which two enzymes, invertase (INV) and glucose dehydrogenase (GDH), are conjugated with peptide linkers. We expected that INV hydrolyzes sucrose into fructose and glucose, then glucose is oxidized at the catalytic subunit of GDH, releasing electrons toward the electrode. It also employed solid binding peptides (SBPs) fused to GDH in chimeric enzyme for the electrical connection of GDH-SBP and electrode. In order to accomplish the desired reaction with chimeric enzyme, we have conducted the following tests; 1) The recombinant plasmid harboring the genetic sequence of two enzymes with an in-between linker was constructed. 2) By production of chimeric proteins, the chain reaction efficiency of fusion constructs was compared depending on the length of the linker. 3) The gold binding peptide (GBP) was fused to sites (N- and C-terminus, or other sites) of the catalytic subunit of GDH. And finally, 4) the electron transfer rates were monitored and compared depending on the GBP tethering sites. Throughout the whole study, we found the factors affecting the occurrence of multienzyme cascade-based bioelectrochemical reactions and how to control these factors to improve the reaction efficiency Figure 1