title: Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics.
abstract: Glucose oxidase is an oxidoreductase exhibiting a high β-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 β-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 β-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.
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany. Electronic address: e.arango@biotec.rwth-aachen.de.
authors.forename: Erik
authors.initials: E
authors.lastname: Arango Gutierrez
authors.forename: Hemanshu
authors.initials: H
authors.lastname: Mundhada
authors.forename: Thomas
authors.initials: T
authors.lastname: Meier
authors.forename: Hartmut
authors.initials: H
authors.lastname: Duefel
authors.forename: Marco
authors.initials: M
authors.lastname: Bocola
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods*; D019020:Directed Molecular Evolution / Q000379:methods; D005947:Glucose / Q000032:analysis* / Q000378:metabolism; D005949:Glucose Oxidase / Q000235:genetics* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D005949:Glucose Oxidase; D005947:Glucose; D010100:Oxygen
keywords: Biosensor; Diabetes; Directed evolution; Glucose oxidase; Oxygen-independent