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 . .

TY  - JOUR
AU  - Tu, Ran
AU  - Martinez, Ronny
AU  - Prodanović, Radivoje
AU  - Klein, Mathias
AU  - Schwaneberg, Ulrich
PY  - 2011
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/1158
AB  - Proteases are industrially important enzymes but often have to be improved for their catalytic efficiency and stabilities to suit applications. Flow cytometry screening technology based on in vitro compartmentalization in double emulsion had been developed and applied on directed evolution of paraoxonase and beta-galactosidase. Further advancements of flow cytometry-based screening technologies will enable an ultra-high throughput of variants offering novel opportunities in directed enzyme evolution under high mutational loads. For the industrially important enzyme class of proteases, a first flow cytometry-based screening system for directed protease evolution has been developed based on an extracellular protease-deficient Bacillus subtilis strain (WB800N), a model protease (subtilisin Carlsberg), and a water-in-oil-in-water double-emulsion technology. B. subtilis WB800N cells are encapsulated in double emulsion with a fluorogenic substrate (rhodamine 110-containing peptide), allowing the screening of protease variants in femtoliter compartments at high throughput. The protease screening technology was validated by employing an epPCR mutant library with a high mutational load and screened for increased resistance toward the inhibitor antipain dihydrochloride. A variant (K127R, T237P, M239I, I269V, Y310F, I372V) with an improved relative resistance was isolated from a small population of active variants, validating the reported protease flow cytometry screening technology for increased inhibitor resistance. (Journal of Biomolecular Screening 2011;16:285-294)
PB  - Sage Publications Inc, Thousand Oaks
T2  - Journal of Biomolecular Screening
T1  - A Flow Cytometry-Based Screening System for Directed Evolution of Proteases
VL  - 16
IS  - 3
SP  - 285
EP  - 294
DO  - 10.1177/1087057110396361
ER  - 

@article{
author = "Tu, Ran and Martinez, Ronny and Prodanović, Radivoje and Klein, Mathias and Schwaneberg, Ulrich",
year = "2011",
abstract = "Proteases are industrially important enzymes but often have to be improved for their catalytic efficiency and stabilities to suit applications. Flow cytometry screening technology based on in vitro compartmentalization in double emulsion had been developed and applied on directed evolution of paraoxonase and beta-galactosidase. Further advancements of flow cytometry-based screening technologies will enable an ultra-high throughput of variants offering novel opportunities in directed enzyme evolution under high mutational loads. For the industrially important enzyme class of proteases, a first flow cytometry-based screening system for directed protease evolution has been developed based on an extracellular protease-deficient Bacillus subtilis strain (WB800N), a model protease (subtilisin Carlsberg), and a water-in-oil-in-water double-emulsion technology. B. subtilis WB800N cells are encapsulated in double emulsion with a fluorogenic substrate (rhodamine 110-containing peptide), allowing the screening of protease variants in femtoliter compartments at high throughput. The protease screening technology was validated by employing an epPCR mutant library with a high mutational load and screened for increased resistance toward the inhibitor antipain dihydrochloride. A variant (K127R, T237P, M239I, I269V, Y310F, I372V) with an improved relative resistance was isolated from a small population of active variants, validating the reported protease flow cytometry screening technology for increased inhibitor resistance. (Journal of Biomolecular Screening 2011;16:285-294)",
publisher = "Sage Publications Inc, Thousand Oaks",
journal = "Journal of Biomolecular Screening",
title = "A Flow Cytometry-Based Screening System for Directed Evolution of Proteases",
volume = "16",
number = "3",
pages = "285-294",
doi = "10.1177/1087057110396361"
}

Tu, R., Martinez, R., Prodanović, R., Klein, M.,& Schwaneberg, U.. (2011). A Flow Cytometry-Based Screening System for Directed Evolution of Proteases. in Journal of Biomolecular Screening
Sage Publications Inc, Thousand Oaks., 16(3), 285-294.
https://doi.org/10.1177/1087057110396361

Tu R, Martinez R, Prodanović R, Klein M, Schwaneberg U. A Flow Cytometry-Based Screening System for Directed Evolution of Proteases. in Journal of Biomolecular Screening. 2011;16(3):285-294.
doi:10.1177/1087057110396361 .

Tu, Ran, Martinez, Ronny, Prodanović, Radivoje, Klein, Mathias, Schwaneberg, Ulrich, "A Flow Cytometry-Based Screening System for Directed Evolution of Proteases" in Journal of Biomolecular Screening, 16, no. 3 (2011):285-294,
https://doi.org/10.1177/1087057110396361 . .