TY  - JOUR
AU  - Ostafe, Raluca
AU  - Prodanović, Radivoje
AU  - Nazor, Jovana
AU  - Fischer, Rainer
PY  - 2014
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/1519
AB  - Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme.
PB  - Cell Press, Cambridge
T2  - Chemistry and Biology
T1  - Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase
VL  - 21
IS  - 3
SP  - 414
EP  - 421
DO  - 10.1016/j.chembiol.2014.01.010
ER  - 

@article{
author = "Ostafe, Raluca and Prodanović, Radivoje and Nazor, Jovana and Fischer, Rainer",
year = "2014",
abstract = "Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme.",
publisher = "Cell Press, Cambridge",
journal = "Chemistry and Biology",
title = "Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase",
volume = "21",
number = "3",
pages = "414-421",
doi = "10.1016/j.chembiol.2014.01.010"
}

Ostafe, R., Prodanović, R., Nazor, J.,& Fischer, R.. (2014). Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase. in Chemistry and Biology
Cell Press, Cambridge., 21(3), 414-421.
https://doi.org/10.1016/j.chembiol.2014.01.010

Ostafe R, Prodanović R, Nazor J, Fischer R. Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase. in Chemistry and Biology. 2014;21(3):414-421.
doi:10.1016/j.chembiol.2014.01.010 .

Ostafe, Raluca, Prodanović, Radivoje, Nazor, Jovana, Fischer, Rainer, "Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase" in Chemistry and Biology, 21, no. 3 (2014):414-421,
https://doi.org/10.1016/j.chembiol.2014.01.010 . .

TY  - 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 . .