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.


Journal

Biosensors & bioelectronics
ISSN: 1873-4235
Titre abrégé: Biosens Bioelectron
Pays: England
ID NLM: 9001289

Informations de publication

Date de publication:
15 Mar 2021
Historique:
received: 30 10 2020
revised: 12 12 2020
accepted: 16 12 2020
pubmed: 10 1 2021
medline: 15 5 2021
entrez: 9 1 2021
Statut: ppublish

Résumé

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).

Identifiants

pubmed: 33421758
pii: S0956-5663(20)30896-4
doi: 10.1016/j.bios.2020.112911
pii:
doi:

Substances chimiques

Blood Glucose 0
Flavin-Adenine Dinucleotide 146-14-5
Heme 42VZT0U6YR
Glucose 1-Dehydrogenase EC 1.1.1.47
Glucose IY9XDZ35W2

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

112911

Informations de copyright

Copyright © 2020 Elsevier B.V. All rights reserved.

Auteurs

Kohei Ito (K)

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.

Junko Okuda-Shimazaki (J)

Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC27599, USA.

Katsuhiro Kojima (K)

Ultizyme International Ltd., 3-9-5 2F, Taihei, Sumida, Tokyo, 130-0011, Japan.

Kazushige Mori (K)

Ultizyme International Ltd., 3-9-5 2F, Taihei, Sumida, Tokyo, 130-0011, Japan.

Wakako Tsugawa (W)

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.

Ryutaro Asano (R)

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.

Kazunori Ikebukuro (K)

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.

Koji Sode (K)

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.

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Classifications MeSH