TY  - JOUR
AU  - Despotovic, Dragana
AU  - Vojcic, Ljubica
AU  - Prodanović, Radivoje
AU  - Martinez, Ronny
AU  - Maurer, Karl-Heinz
AU  - Schwaneberg, Ulrich
PY  - 2012
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/1308
AB  - Directed evolution offers opportunities to improve promiscuous activities of hydrolases in rounds of diversity generation and high-throughput screening. In this article, we developed and validated a screening platform to improve the perhydrolytic activity of proteases and likely other hydrolases (e.g., lipases or esterases). Key was the development of a highly sensitive fluorescent assay (sensitivity in the mu M range) based on 3-carboxy-7-hydroxycoumarin (HCC) formation. HCC is released through an hypobromite-mediated oxidation of 7-(4'-aminophenoxy)-3-carboxycoumarin (APCC), which enables for the first time a continuous measurement of peroxycarboxylic acid formation with a standard deviation of 11% in microtiter plates with a wide pH range window (5-9). As example, subtilisin Carlsberg was subjected to site saturation mutagenesis at position G165, yielding a variant T58A/G165L/L216W with 5.4-fold increased k(cat) for perhydrolytic activity compared with wild type.
PB  - Sage Publications Inc, Thousand Oaks
T2  - Journal of Biomolecular Screening
T1  - Fluorescent Assay for Directed Evolution of Perhydrolases
VL  - 17
IS  - 6
SP  - 796
EP  - 805
DO  - 10.1177/1087057112438464
ER  - 

@article{
author = "Despotovic, Dragana and Vojcic, Ljubica and Prodanović, Radivoje and Martinez, Ronny and Maurer, Karl-Heinz and Schwaneberg, Ulrich",
year = "2012",
abstract = "Directed evolution offers opportunities to improve promiscuous activities of hydrolases in rounds of diversity generation and high-throughput screening. In this article, we developed and validated a screening platform to improve the perhydrolytic activity of proteases and likely other hydrolases (e.g., lipases or esterases). Key was the development of a highly sensitive fluorescent assay (sensitivity in the mu M range) based on 3-carboxy-7-hydroxycoumarin (HCC) formation. HCC is released through an hypobromite-mediated oxidation of 7-(4'-aminophenoxy)-3-carboxycoumarin (APCC), which enables for the first time a continuous measurement of peroxycarboxylic acid formation with a standard deviation of 11% in microtiter plates with a wide pH range window (5-9). As example, subtilisin Carlsberg was subjected to site saturation mutagenesis at position G165, yielding a variant T58A/G165L/L216W with 5.4-fold increased k(cat) for perhydrolytic activity compared with wild type.",
publisher = "Sage Publications Inc, Thousand Oaks",
journal = "Journal of Biomolecular Screening",
title = "Fluorescent Assay for Directed Evolution of Perhydrolases",
volume = "17",
number = "6",
pages = "796-805",
doi = "10.1177/1087057112438464"
}

Despotovic, D., Vojcic, L., Prodanović, R., Martinez, R., Maurer, K.,& Schwaneberg, U.. (2012). Fluorescent Assay for Directed Evolution of Perhydrolases. in Journal of Biomolecular Screening
Sage Publications Inc, Thousand Oaks., 17(6), 796-805.
https://doi.org/10.1177/1087057112438464

Despotovic D, Vojcic L, Prodanović R, Martinez R, Maurer K, Schwaneberg U. Fluorescent Assay for Directed Evolution of Perhydrolases. in Journal of Biomolecular Screening. 2012;17(6):796-805.
doi:10.1177/1087057112438464 .

Despotovic, Dragana, Vojcic, Ljubica, Prodanović, Radivoje, Martinez, Ronny, Maurer, Karl-Heinz, Schwaneberg, Ulrich, "Fluorescent Assay for Directed Evolution of Perhydrolases" in Journal of Biomolecular Screening, 17, no. 6 (2012):796-805,
https://doi.org/10.1177/1087057112438464 . .