How to engineer glucose oxidase for mediated electron transfer. Issue 10 (25th July 2018)

• Record Type: Journal Article
• Authors: Arango Gutierrez, Erik; Wallraf, Anne‐Maria; Balaceanu, Alexandra; Bocola, Marco; Davari, Mehdi D.; Meier, Thomas; Duefel, Hartmut; Schwaneberg, Ulrich
• Is Part Of: Biotechnology and bioengineering. Volume 115:Issue 10(2018)
• Page Start: 2405
• Page End: 2415
• Publication Date: 2018-07-25
• Subjects: directed evolution -- electron mediators -- glucose oxidase (GOx) -- Marcus theory -- molecular docking -- protein engineering; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.26785
• Languages: English
• ISSNs: 0006-3592

Abstract

Abstract: Glucose oxidase (GOx) is of high industrial interest for glucose sensing because of its high β‐d ‐glucose specificity. The efficient and specific electrochemical communication between the redox center and electrodes is crucial to ensure accurate glucose determination. The efficiency of the electron transfer rates (ETR) with GOx, together with quinone diamine based mediators, is low and differs even among mediator derivatives. To design optimized enzyme–mediator couples and to describe a mediator binding model, a joint experimental and computational study was performed based on an oxygen‐independent GOx variant V7 and two quinone diimine based electron mediators (QDM‐1 and QDM‐2), which differ in polarity and size, and ferrocenemethanol (FM). A site saturation library at position 414 was screened with all three mediators and yielded four beneficial substitutions Tyr, Met, Leu, and Val. The variants showed increased mediator activity for the more polar QDM‐2 with a simultaneously decreased activity for the less polar and smaller QDM‐1 and for FM. The variant GOx V7‐I414Y exhibited the biggest change for the quinone diimine derivatives compared with V7 (QDM‐1: 55.9 U/mg V7, 33.2 U/mg V7‐I414Y; QDM‐2: 2.7 U/mg V7, 12.9 U/mg V7‐I414Y). Theoretical ETR calculated based on the Marcus theory were in good agreement with the experimental results. Molecular docking studies revealed a preferable binding of the two QD mediators directly in the active site, 3.5 Å away from the N5 atom of the flavin adenine dinucleotide (FAD) and in direct vicinity to position 414. In summary, position 414 in the active site was identified to modulate the electron shuttling from the FAD of the GOx to small water‐soluble mediators dependent on the polarity and size of residue 414 and on the polarity and size of the mediator. The presented mediator binding model offers a promising possibility for the design of optimized enzyme–mediator couples. Abstract : A joint protein engineering and computational study of glucose oxidase (GOx) for the development of improved glucose biosensing devices was performed. Position 414 in the active site was identified to modulate the electron shuttling from the FAD of the GOx to small water‐soluble mediators dependent on the polarity and size of residue 414 and the mediator. The mediator binding model offers a promising possibility for the design of optimized enzyme–mediator couples.