Directed evolution of glucose oxidase from Aspergillus niger for ferrocenemethanol-mediated electron transfer
Само за регистроване кориснике
2007
Чланак у часопису (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
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.
Кључне речи:
Biofuel cell / Directed evolution / Ferrocenemethanol / Glucose oxidase / MediatorИзвор:
Biotechnology Journal, 2007, 2, 2, 241-248Колекције
Институција/група
Hemijski fakultet / Faculty of ChemistryTY - JOUR AU - Zhu, Z. AU - Wang, M. AU - Gautam, A. AU - Nazor, Jovana AU - Momeu, C. AU - Prodanović, Radivoje AU - Schwaneberg, U. PY - 2007 UR - https://cherry.chem.bg.ac.rs/handle/123456789/77 AB - 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. T2 - Biotechnology Journal T1 - Directed evolution of glucose oxidase from Aspergillus niger for ferrocenemethanol-mediated electron transfer VL - 2 IS - 2 SP - 241 EP - 248 DO - 10.1002/biot.200600185 ER -
@article{ author = "Zhu, Z. and Wang, M. and Gautam, A. and Nazor, Jovana and Momeu, C. and Prodanović, Radivoje and Schwaneberg, U.", year = "2007", abstract = "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.", journal = "Biotechnology Journal", title = "Directed evolution of glucose oxidase from Aspergillus niger for ferrocenemethanol-mediated electron transfer", volume = "2", number = "2", pages = "241-248", doi = "10.1002/biot.200600185" }
Zhu, Z., Wang, M., Gautam, A., Nazor, J., Momeu, C., Prodanović, R.,& Schwaneberg, U.. (2007). Directed evolution of glucose oxidase from Aspergillus niger for ferrocenemethanol-mediated electron transfer. in Biotechnology Journal, 2(2), 241-248. https://doi.org/10.1002/biot.200600185
Zhu Z, Wang M, Gautam A, Nazor J, Momeu C, Prodanović R, Schwaneberg U. Directed evolution of glucose oxidase from Aspergillus niger for ferrocenemethanol-mediated electron transfer. in Biotechnology Journal. 2007;2(2):241-248. doi:10.1002/biot.200600185 .
Zhu, Z., Wang, M., Gautam, A., Nazor, Jovana, Momeu, C., Prodanović, Radivoje, Schwaneberg, U., "Directed evolution of glucose oxidase from Aspergillus niger for ferrocenemethanol-mediated electron transfer" in Biotechnology Journal, 2, no. 2 (2007):241-248, https://doi.org/10.1002/biot.200600185 . .