Making glucose oxidase fit for biofuel cell applications by directed protein evolution

Progress in miniature chip-design raises demands for implantable power sources in health care applications such as continuous glucose monitoring of diabetic patients. Pioneered by Adam Heller, miniaturized enzymatic biofuel cells (mBCs) convert blood sugars into electrical energy by employing for example glucose oxidase (GOx) on the anode and bilirubin oxidase on the cathode. To match application demands it is crucial to increase lifetime and power output of mBCs. The power output has been limited by the performance of GOx on the anode. We developed a glucose oxidase detection assay (GODA) as medium-throughput screening system for improving GOx properties by directed protein evolution. GODA is a reaction product detection assay based on coupled enzymatic reactions leading to NADPH formation which is recorded at 340 nm. The main advantage of the assay is that it detects the production of d-gluconolactone instead of the side-product hydrogen peroxide and enables to improve bioelectrochemical properties of GOx. For validating the screening system, a mutagenic library of GOx from Aspergillus niger (EC 1.1.3.4) was generated and screened for improved activity using Saccharomyces cerevisiae as host. Directed evolution resulted in a GOx mutant I115V with 1.4–1.5-fold improved activity for β-d-glucose (Vmax from 7.94 to 10.81 μmol min−1 mg−1; Km ∼ 19–21 mM) and oxygen consumption kinetics correlate well [Vmax (O2) from 5.94 to 8.34 μmol min−1 mg−1; Km (O2) from 700 to 474 μM]. The developed mutagenic protocol and GODA represent a proof-of-principle that GOx can be evolved by directed evolution in S. cerevisiae for putative use in biofuel cells.