Fluorescent Assay for Directed Evolution of Perhydrolases
Само за регистроване кориснике
2012
Аутори
Despotovic, DraganaVojcic, Ljubica
Prodanović, Radivoje
Martinez, Ronny
Maurer, Karl-Heinz
Schwaneberg, Ulrich
Чланак у часопису (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
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.
Кључне речи:
high-throughput screening / hydrolase / perhydrolysis / peroxycarboxylic acid / subtilisin CarlsbergИзвор:
Journal of Biomolecular Screening, 2012, 17, 6, 796-805Издавач:
- Sage Publications Inc, Thousand Oaks
Финансирање / пројекти:
- Henkel AG Co. KGaA
- German government through the Bundesministerium fur Bildung and Forschung [Bioindustrie-2021, FKZ0315250]
Напомена:
- Free full text: https://doi.org/10.1177/1087057112438464
DOI: 10.1177/1087057112438464
ISSN: 1087-0571
PubMed: 22392808
WoS: 000305246000009
Scopus: 2-s2.0-84862234568
Колекције
Институција/група
Hemijski fakultet / Faculty of ChemistryTY - 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 . .