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 MnCl(2)) and Saccharomyces cerevisiae was employed as host for secreted GOx expression. Two screening of approximately 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 degrees C (wild type, WT) to 62 degrees C (T30S I94V) and pH stability was improved at basic pH (pH 8-11). K(m) for glucose remained nearly unchanged (20.8 mM WT; 21.3 mM T30S I94V) and k(cat) increased (69.5/s WT; 137.7/s T30S I94V).
Zhu
Ziwei
Z
International University Bremen, Bremen, Germany.
Wang
Mian
M
Gautam
Amit
A
Nazor
Jovana
J
Momeu
Carmen
C
Prodanovic
Radivoje
R
Schwaneberg
Ulrich
U
D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D004579:Electron Transport; D004795:Enzyme Stability; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D005949:Glucose Oxidase / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013696:Temperature
D005296:Ferrous Compounds; D005656:Fungal Proteins; D011994:Recombinant Proteins; C071550:ferrocenemethanol; D005949:Glucose Oxidase; D005947:Glucose