Optimization of Glucose oxidase towards oxygen independency and high mediator activity for amperometric glucose determination in diabetes analytics

摘要

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, and one simultaneous site saturation library (OmniChange; 4 positions) was performed in the first project part. A for diabetes care well suited mediator (quinone diimine) was selected and the GOx variant (T30V I94V) served as starting point. Two iterative rounds of random diversity generation and screening yielded 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 accomplish the main challenge in diabetes analytics by combining high glucose specificity with oxygen independency. In the second project part, the influence of residue 414 on the interaction of small soluble mediator compounds and GOx were investigated. A site saturation experiment revealed a subset of amino acids (Y, M, L, V) which change the preference of GOx from the QDM-1 to the more polar and sterically more demanding QDM-2. The substitution I414Y resulted in a 4.8-fold increased activity of V7 employing the QDM-2 system, what was traced back to a higher affinity through the change to a more polar residue and structural reorganization. Furthermore, it was shown that substitutions of residue 414 also influence the activity employing ferrocenemethanol, a prominent mediator example for GOx. Molecular docking studies revealed that small water soluble mediator compounds bind directly in the active site of GOx in close vicinity to residue 414. First time an enzyme was adapted to a specific mediator component by protein engineering. This approach might not only play an important role in diagnostics but also in the field of bio-fuel cells in order to adopt electron donor or acceptor enzymes to electrode systems. In the last project part a bio-process was established in order to ensure a fast and efficient production of GOx for characterization studies. Media screening and investigations of the feeding strategy revealed a batch fermentation process with a process time of 48 h. The fermentation process was characterized with respect to bio-mass formation, glucose consumption, respiratory properties (RQ, pO2), acetate formation and GOx formation. A downstream process was scaled-up for the handling of 10 to 20 L of cell broth. Tangential flow filtration was introduced for the cell harvest (micro-filtration) and product isolation (ultra-filtration) in order to handle the respective volumes. The optimization of an anion-exchange chromatography step resulted in a downstream process with only one purification step. Samples with a GOx-content higher 90 % could be reached.