Site-Directed Mutagenesis of Multicopper Oxidase from Hyperthermophilic Archaea for High-Voltage Biofuel Cells
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Site-Directed Mutagenesis of Multicopper Oxidase from Hyperthermophilic Archaea for High-Voltage Biofuel Cells Eiichiro Takamura 1 & Shunsuke Taki 1 & Hiroaki Sakamoto 1 & Takenori Satomura 2 & Haruhiko Sakuraba 3 & Toshihisa Ohshima 4 & Shin-ichiro Suye 1,2 Received: 15 April 2020 / Accepted: 29 September 2020/ # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Enzymes from hyperthermophilic archaea are potential candidates for industrial use because of their superior pH, thermal, and long-term stability, and are expected to improve the long-term stability of biofuel cells (BFCs). However, the reported multicopper oxidase (MCO) from hyperthermophilic archaea has lower redox potential than MCOs from other organisms, which leads to a decrease in the cell voltage of BFCs. In this study, we attempted to positively shift the redox potential of the MCO from hyperthermophilic archaeon Pyrobaculum aerophilum (McoP). Mutations (M470L and M470F) were introduced into the axial ligand of the T1 copper atom of McoP, and the enzymatic chemistry and redox potentials were compared with that of the parent (M470). The redox potentials of M470L and M470F shifted positively by about 0.07 V compared with that of M470. In addition, the catalytic activity of the mutants towards 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) increased 1.2–1.3-fold. The thermal stability of the mutants and the electrocatalytic performance for O2 reduction of M470F was slightly reduced compared with that of M470. This research provides useful enzymes for application as biocathode catalysts for high-voltage BFCs. Keywords Site-directed mutagenesis . Multicopper oxidase . Redox potential . Biocathode . Biofuel cell
Introduction In recent times, biofuel cells (BFCs) have attracted attention as a potential alternative energy source owing to the need for safe and renewable energy. BFCs generate electricity using
* Eiichiro Takamura e_takamr@u–fukui.ac.jp Extended author information available on the last page of the article
Applied Biochemistry and Biotechnology
substances such as sugar, alcohol, and amino acids as fuel with enzymes as biocatalyst [1–3]. They can be potentially applied as a power source for batteries of mobile devices and implanted devices due to their many advantages, i.e., operation in mild conditions, ease of miniaturization, safety [4, 5]. However, practical applications of BFCs are limited by the low long-term stability of enzymes and low power density. We have been investigating biocathodes using multicopper oxidase (MCO) from hyperthermophilic archaea Pyrobaculum aerophilum (McoP) and their application for BFCs, because MCOs from hyperthermophilic archaea show superior thermostability and long-term stability when compared with enzymes from many other organisms [6, 7]. Previously, we reported mediated electron transfer (MET)–type BFCs combined with McoP and pyrroloquinoline quinone–dependent glucose dehydrogenase (PQQGDH) [8]; it retained 70% of its power density after 14 days [8]. However,
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