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News

Lessons from diversity of directed evolution experiments by an analysis of 3,000 mutations.
14 July 2014
Zhao J, Kardashliev T, Joelle
Biotechnol Bioeng 2014
Diversity generation by random mutagenesis is often the first key step in directed evolution...
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Bacillus gibsonii alkaline protease
1 November 2012
Martinez R, Jakob F, Tu R, Sie
Biotechnol Bioeng. 2013 Mar
Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting...
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Review: To get what we aim for - progress in diversity generation methods
5 June 2013
Ruff AJ, Dennig A, Schwaneberg
FEBS J., 280 (2013), 2961-2978
Protein re-engineering by directed evolution has become a standard approach for tailoring enzymes...
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Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics
20 June 2013
Arango Gutierrez E, Mundhada H
Biosensors and Bioelectronics
Glucose oxidase is an oxidoreductase exhibiting a high β-d-glucose specificity and high stability...
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Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics

Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics
20 June 2013
Arango Gutierrez E, Mundhada H, Meier T, Duefel H, Bocola M, Schwaneberg U
Biosensors and Bioelectronics 50 (2013) 84–90

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: 7.4U/mg WT, 47.5U/mg V7; oxygen activity: 172.3U/mg WT, 30.1U/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.