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  - Lee, Wook
AU  - Vojcic, Ljubica
AU  - Despotovic, Dragana
AU  - Prodanović, Radivoje
AU  - Maurer, Karl-Heinz
AU  - Schwaneberg, Ulrich
AU  - Zacharias, Martin
PY  - 2010
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/1039
AB  - The perhydrolysis reaction in hydrolases is an important example of catalytic promiscuity and has many potential industrial applications. The mechanisms of perhydrolase activity of a subtilisin Carlsberg mutant and of an aryl-esterase mutant have been investigated using classical molecular dynamics simulations of the second tetrahedral intermediate (TI) state. The simulations demonstrated that hydrogen bonding between the second TI of the perhydrolysis reaction is possible in the mutants but not wild type. The stabilization by hydrogen bonds was specific for the perhydrolysis intermediate and either no hydrogen bonding or only weakened hydrogen bonding to the second TI state of the hydrolysis reaction was observed. Furthermore, a significant hindrance to the formation of the catalytically important hydrogen bond between His64 and Ser221 in the catalytic triad by competing hydrogen bonds was found for the subtilisin mutant but not wild type enzyme in case of the hydrolysis intermediate. The opposite was observed in case of the perhydrolysis intermediate. The result offers a qualitative explanation for the overall reduced hydrolysis activity of the subtilisin mutant. In addition, the simulations also explain qualitatively the perhydrolysis activity of the enzyme variants and may be helpful for designing enzyme mutants with further improved perhydrolysis activity.
PB  - Springer, New York
T2  - Theoretical Chemistry Accounts
T1  - Rationalizing perhydrolase activity of aryl-esterase and subtilisin Carlsberg mutants by molecular dynamics simulations of the second tetrahedral intermediate state
VL  - 125
IS  - 3-6
SP  - 375
EP  - 386
DO  - 10.1007/s00214-009-0611-3
ER  - 

@article{
author = "Lee, Wook and Vojcic, Ljubica and Despotovic, Dragana and Prodanović, Radivoje and Maurer, Karl-Heinz and Schwaneberg, Ulrich and Zacharias, Martin",
year = "2010",
abstract = "The perhydrolysis reaction in hydrolases is an important example of catalytic promiscuity and has many potential industrial applications. The mechanisms of perhydrolase activity of a subtilisin Carlsberg mutant and of an aryl-esterase mutant have been investigated using classical molecular dynamics simulations of the second tetrahedral intermediate (TI) state. The simulations demonstrated that hydrogen bonding between the second TI of the perhydrolysis reaction is possible in the mutants but not wild type. The stabilization by hydrogen bonds was specific for the perhydrolysis intermediate and either no hydrogen bonding or only weakened hydrogen bonding to the second TI state of the hydrolysis reaction was observed. Furthermore, a significant hindrance to the formation of the catalytically important hydrogen bond between His64 and Ser221 in the catalytic triad by competing hydrogen bonds was found for the subtilisin mutant but not wild type enzyme in case of the hydrolysis intermediate. The opposite was observed in case of the perhydrolysis intermediate. The result offers a qualitative explanation for the overall reduced hydrolysis activity of the subtilisin mutant. In addition, the simulations also explain qualitatively the perhydrolysis activity of the enzyme variants and may be helpful for designing enzyme mutants with further improved perhydrolysis activity.",
publisher = "Springer, New York",
journal = "Theoretical Chemistry Accounts",
title = "Rationalizing perhydrolase activity of aryl-esterase and subtilisin Carlsberg mutants by molecular dynamics simulations of the second tetrahedral intermediate state",
volume = "125",
number = "3-6",
pages = "375-386",
doi = "10.1007/s00214-009-0611-3"
}

Lee, W., Vojcic, L., Despotovic, D., Prodanović, R., Maurer, K., Schwaneberg, U.,& Zacharias, M.. (2010). Rationalizing perhydrolase activity of aryl-esterase and subtilisin Carlsberg mutants by molecular dynamics simulations of the second tetrahedral intermediate state. in Theoretical Chemistry Accounts
Springer, New York., 125(3-6), 375-386.
https://doi.org/10.1007/s00214-009-0611-3

Lee W, Vojcic L, Despotovic D, Prodanović R, Maurer K, Schwaneberg U, Zacharias M. Rationalizing perhydrolase activity of aryl-esterase and subtilisin Carlsberg mutants by molecular dynamics simulations of the second tetrahedral intermediate state. in Theoretical Chemistry Accounts. 2010;125(3-6):375-386.
doi:10.1007/s00214-009-0611-3 .

Lee, Wook, Vojcic, Ljubica, Despotovic, Dragana, Prodanović, Radivoje, Maurer, Karl-Heinz, Schwaneberg, Ulrich, Zacharias, Martin, "Rationalizing perhydrolase activity of aryl-esterase and subtilisin Carlsberg mutants by molecular dynamics simulations of the second tetrahedral intermediate state" in Theoretical Chemistry Accounts, 125, no. 3-6 (2010):375-386,
https://doi.org/10.1007/s00214-009-0611-3 . .