Directed evolution of glucose oxidase from Aspergillus niger for ferrocenemethanol-mediated electron transfer

A directed evolution protocol was developed for glucose oxidase (GOx) from Aspergillus niger that mimics applications conditions and employs a well-known mediator, oxidized ferrocenemethanol, in a medium throughput screen (96-well plate format). Upon reduction, oxidized ferrocenemethanol shows a color change from blue to pale yellow that can be recorded at 625 nm. Under optimized screening conditions, a CV of less than 20% was achieved in 96-well microtiter plates. For validating the screening system, two mutant libraries of GOx were generated by standard error-prone PCR conditions (0.04 mM MnCl2) and Saccharomyces cerevisiae was employed as host for secreted GOx expression. Two screening of ∼2000 GOx mutants yielded a double mutant (T30S I94V) with improved pH and thermal resistance. Thermal resistance at a residual activity of 50% was increased from 58°C (wild type, WT) to 62°C (T30S I94V) and pH stability was improved at basic pH (pH 8-11). Km for glucose remained nearly unchanged (20.8 mM WT; 21.3 mM T30S I94V) and kcat increased (69.5/s WT; 137.7/s T30S 194V). © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim