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Ultrahigh Throughput Screening System for Directed Glucose Oxidase Evolution in Yeast Cells

Prodanović, Radivoje; Ostafe, Raluca; Scacioc, Andreea; Schwaneberg, Ulrich

(Bentham Science Publ Ltd, Sharjah, 2011)

TY  - JOUR
AU  - Prodanović, Radivoje
AU  - Ostafe, Raluca
AU  - Scacioc, Andreea
AU  - Schwaneberg, Ulrich
PY  - 2011
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/1142
AB  - A compartmentalized tyramide labeling system (CoaTi) employing flow cytometry for sorting of yeast cells was developed as ultrahigh throughput screening for Glucose oxidase (GOx) from Aspergillus niger. CoaTi combines in vitro compartmentalization technology with the CARD reporter system which uses fluorescein tyramide labels for detection of peroxidase activity. Physical connection between cells and fluorescein tyramide radicals was achieved by compartmentalization of yeast cells inside microdroplets of single water-in-oil emulsions. After reaction cells were recovered from single emulsions and sorted by flow cytometry, an error prone PCR mutant library of Glucose oxidase (GOx) containing 10(7) cells and similar to 10(5) of different GOx variants was screened. Mutagenic conditions of GOx mutant library were selected to generate  lt 1% of active GOx population in order to explore influence of high mutation frequency on GOx activity. GOx variant Mut12 that contains 5 mutations (N2Y, K13E, T30V, I94V, K152R) showed a 1.2 times decreased K-m (22.0 vs 18.1 mM) and a 2.7 fold increased k(cat) (150 s(-1) vs 54.8 s(-1)) compared to wt GOx. Compared to the employed parent B11 GOx (16 mM, 80 s(-1)) it has a slightly increased K-m and 1.8 times increased k(cat).
PB  - Bentham Science Publ Ltd, Sharjah
T2  - Combinatorial Chemistry and High Throughput Screening
T1  - Ultrahigh Throughput Screening System for Directed Glucose Oxidase Evolution in Yeast Cells
VL  - 14
IS  - 1
SP  - 55
EP  - 60
DO  - 10.2174/1386207311107010055
ER  - 
@article{
author = "Prodanović, Radivoje and Ostafe, Raluca and Scacioc, Andreea and Schwaneberg, Ulrich",
year = "2011",
url = "http://cherry.chem.bg.ac.rs/handle/123456789/1142",
abstract = "A compartmentalized tyramide labeling system (CoaTi) employing flow cytometry for sorting of yeast cells was developed as ultrahigh throughput screening for Glucose oxidase (GOx) from Aspergillus niger. CoaTi combines in vitro compartmentalization technology with the CARD reporter system which uses fluorescein tyramide labels for detection of peroxidase activity. Physical connection between cells and fluorescein tyramide radicals was achieved by compartmentalization of yeast cells inside microdroplets of single water-in-oil emulsions. After reaction cells were recovered from single emulsions and sorted by flow cytometry, an error prone PCR mutant library of Glucose oxidase (GOx) containing 10(7) cells and similar to 10(5) of different GOx variants was screened. Mutagenic conditions of GOx mutant library were selected to generate  lt 1% of active GOx population in order to explore influence of high mutation frequency on GOx activity. GOx variant Mut12 that contains 5 mutations (N2Y, K13E, T30V, I94V, K152R) showed a 1.2 times decreased K-m (22.0 vs 18.1 mM) and a 2.7 fold increased k(cat) (150 s(-1) vs 54.8 s(-1)) compared to wt GOx. Compared to the employed parent B11 GOx (16 mM, 80 s(-1)) it has a slightly increased K-m and 1.8 times increased k(cat).",
publisher = "Bentham Science Publ Ltd, Sharjah",
journal = "Combinatorial Chemistry and High Throughput Screening",
title = "Ultrahigh Throughput Screening System for Directed Glucose Oxidase Evolution in Yeast Cells",
volume = "14",
number = "1",
pages = "55-60",
doi = "10.2174/1386207311107010055"
}
Prodanović, R., Ostafe, R., Scacioc, A.,& Schwaneberg, U. (2011). Ultrahigh Throughput Screening System for Directed Glucose Oxidase Evolution in Yeast Cells.
Combinatorial Chemistry and High Throughput Screening
Bentham Science Publ Ltd, Sharjah., 14(1), 55-60.
https://doi.org/10.2174/1386207311107010055
Prodanović R, Ostafe R, Scacioc A, Schwaneberg U. Ultrahigh Throughput Screening System for Directed Glucose Oxidase Evolution in Yeast Cells. Combinatorial Chemistry and High Throughput Screening. 2011;14(1):55-60
Prodanović Radivoje, Ostafe Raluca, Scacioc Andreea, Schwaneberg Ulrich, "Ultrahigh Throughput Screening System for Directed Glucose Oxidase Evolution in Yeast Cells" Combinatorial Chemistry and High Throughput Screening, 14, no. 1 (2011):55-60,
https://doi.org/10.2174/1386207311107010055 .
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