How to engineer glucose oxidase for mediated electron transfer.

Glucose oxidase (GOx) is of high industrial interest for glucose sensing because of its high ß-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.

Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics.

Glucose oxidase is an oxidoreductase exhibiting a high ß-D-glucose specificity and high stability which renders glucose oxidase well-suited for applications in diabetes care. Nevertheless, GOx activity is highly oxygen dependent which can lead to inaccuracies in amperometric ß-D-glucose determinations. Therefore a directed evolution campaign with two rounds of random mutagenesis (SeSaM followed by epPCR), site saturation mutagenesis studies on individual positions, and one simultaneous site saturation library (OmniChange; 4 positions) was performed. A diabetes care well suited mediator (quinone diimine) was selected and the GOx variant (T30V I94V) served as starting point. For directed GOx evolution a microtiter plate detection system based on the quinone diimine mediator was developed and the well-known ABTS-assay was applied in microtiter plate format to validate oxygen independency of improved GOx variants. Two iterative rounds of random diversity generation and screening yielded to two subsets of amino acid positions which mainly improved activity (A173, A332) and oxygen independency (F414, V560). Simultaneous site saturation of all four positions with a reduced subset of amino acids using the OmniChange method yielded finally variant V7 with a 37-fold decreased oxygen dependency (mediator activity: 7.4 U/mg WT, 47.5 U/mg V7; oxygen activity: 172.3 U/mg WT, 30.1 U/mg V7). V7 is still highly ß-D-glucose specific, highly active with the quinone diimine mediator and thermal resistance is retained (prerequisite for GOx coating of diabetes test stripes). The latter properties and V7's oxygen insensitivity make V7 a very promising candidate to replace standard GOx in diabetes care applications.