Strategic design and improvement of the internal electron transfer of heme b domain-fused glucose dehydrogenase for use in direct electron transfer-type glucose sensors

Kohei Ito; Junko Okuda-Shimazaki; Katsuhiro Kojima; Kazushige Mori; Wakako Tsugawa; Ryutaro Asano; Kazunori Ikebukuro; Koji Sode

doi:10.1016/j.bios.2020.112911

Strategic design and improvement of the internal electron transfer of heme b domain-fused glucose dehydrogenase for use in direct electron transfer-type glucose sensors

Biosens Bioelectron. 2021 Mar 15:176:112911. doi: 10.1016/j.bios.2020.112911. Epub 2020 Dec 17.

Authors

Kohei Ito¹, Junko Okuda-Shimazaki², Katsuhiro Kojima³, Kazushige Mori³, Wakako Tsugawa¹, Ryutaro Asano¹, Kazunori Ikebukuro¹, Koji Sode⁴

Affiliations

¹ Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan.
² Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC27599, USA.
³ Ultizyme International Ltd., 3-9-5 2F, Taihei, Sumida, Tokyo, 130-0011, Japan.
⁴ Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC27599, USA. Electronic address: ksode@email.unc.edu.

PMID: 33421758
DOI: 10.1016/j.bios.2020.112911

Abstract

A fusion enzyme composed of an Aspergillus flavus-derived flavin adenine dinucleotide glucose dehydrogenase (AfGDH) and an electron transfer domain of Phanerochaete chrysosporium-derived cellobiose dehydrogenase (Pcyb) was previously reported to show the direct electron transfer (DET) ability to an electrode. However, its slow intramolecular electron transfer (IET) rate from the FAD to the heme, limited the sensor signals. In this study, fusion FADGDH (Pcyb-AfGDH) enzymes were strategically redesigned by performing docking simulation, following surface-electrostatic potential estimation in the predicted area. Based on these predictions, we selected the amino acid substitution on Glu324, or on Asn408 to Lys to increase the positive charge at the rim of the interdomain region. Pcyb-AfGDH mutants were recombinantly produced using Pichia pastoris as the host microorganism, and their IET was evaluated. Spectroscopic observations showed that the Glu324Lys (E324K) and Asn408Lys (N408K) Pcyb-AfGDH mutants showed approximately 1.70- and 9.0-fold faster IET than that of wildtype Pcyb-AfGDH, respectively. Electrochemical evaluation revealed that the mutant Pcyb-AfGDH-immobilized electrodes showed higher DET current values than that of the wildtype Pcyb-AfGDH-immobilized electrodes at pH 6.5, which was approximately 9-fold higher in the E324K mutant and 15-fold higher in the N408K mutant, than in the wildtype. Glucose enzyme sensors employing N408K mutant was able to measure glucose concentration under physiological condition using artificial interstitial fluid at pH 7.4, whereas the one with wildtype Pcyb-AfGDH was not. These results indicated that the sensor employed the redesigned mutant Pcyb-AfGDH can be used for future continuous glucose monitoring system based on direct electron transfer principle. (247 words).

Keywords: Direct electron transfer; Docking simulation; Fusion protein; Glucose dehydrogenase; Surface electrostatics.

MeSH terms

Biosensing Techniques*
Blood Glucose
Blood Glucose Self-Monitoring
Electron Transport
Electrons
Flavin-Adenine Dinucleotide / metabolism
Glucose
Glucose 1-Dehydrogenase* / metabolism
Heme
Saccharomycetales

Substances

Blood Glucose
Flavin-Adenine Dinucleotide
Heme
Glucose 1-Dehydrogenase
Glucose

Supplementary concepts

Komagataella pastoris