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.
Direct electron transfer
Docking simulation
Fusion protein
Glucose dehydrogenase
Surface electrostatics
Journal
Biosensors & bioelectronics
ISSN: 1873-4235
Titre abrégé: Biosens Bioelectron
Pays: England
ID NLM: 9001289
Informations de publication
Date de publication:
15 Mar 2021
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
112911Informations de copyright
Copyright © 2020 Elsevier B.V. All rights reserved.