caa a22 4500 605500487 CHVBK 20210128100556.0 cr unu---uuuuu 210128e20150601xx s 000 0 eng 10.1007/s00253-015-6612-3 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6612-3 A first continuous 4-aminoantipyrine (4-AAP)-based screening system for directed esterase evolution [Elektronische Daten] [Nina Lülsdorf, Ljubica Vojcic, Hendrik Hellmuth, Thomas Weber, Nina Mußmann, Ronny Martinez, Ulrich Schwaneberg] Esterases hydrolyze ester bonds with an often high stereoselectivity as well as regioselectivity and are therefore industrially employed in the synthesis of pharmaceuticals, in food processing, and in laundry detergents. Continuous screening systems based on p-nitrophenyl- (e.g., p-nitrophenyl acetate) or umbelliferyl-esters are commonly used in directed esterase evolution campaigns. Ongoing challenges in directed esterase evolution are screening formats which offer a broad substrate spectrum, especially for complex aromatic substrates. In this report, a novel continuous high throughput screening system for indirect monitoring of esterolytic activity was developed and validated by detection of phenols employing phenyl benzoate as substrate and p-nitrobenzyl esterase (pNBEBL from Bacillus licheniformis) as catalyst. The released phenol directly reacts with 4-aminoantipyrine yielding the red compound 1,5-dimethyl-4-(4-oxo-cyclohexa-2,5-dienylidenamino)-2-phenyl-1,2-dihydro-pyrazol-3-one. In this continuous B. licheniformis esterase activity detection system (cBLE-4AAP), the product formation is followed through an increase in absorbance at 509nm. The cBLE-4AAP screening system was optimized in 96-well microtiter plate format in respect to standard deviation (5%), linear detection range (15 to 250μM), lower detection limit (15μM), and pH (7.4 to 10.4). The cBLE-4AAP screening system was validated by screening a random epPCR pNBEBL mutagenesis library (2000 clones) for improved esterase activity at elevated temperatures. Finally, the variant T3 (Ser378Pro) was identified which nearly retains its specific activity at room temperature (WT 1036U/mg and T3 929U/mg) and shows compared to WT a 4.7-fold improved residual activity after thermal treatment (30 min incubation at 69.4°C; WT 170U/mg to T3 804U/mg). Springer-Verlag Berlin Heidelberg, 2015 Directed evolution nationallicence High-throughput screening nationallicence Microtiter plate nationallicence Esterase nationallicence Thermal resistance nationallicence Lülsdorf Nina DWI-Leibniz Institut für Interaktive Materialien, Forckenbeckstraße 50, 52056, Aachen, Germany aut Vojcic Ljubica Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany aut Hellmuth Hendrik International Research Laundry and Home Care, Biotechnology, Henkel AG & Co. KGaA, 40191, Düsseldorf, Germany aut Weber Thomas International Research Laundry and Home Care, Biotechnology, Henkel AG & Co. KGaA, 40191, Düsseldorf, Germany aut Mußmann Nina International Research Laundry and Home Care, Biotechnology, Henkel AG & Co. KGaA, 40191, Düsseldorf, Germany aut Martinez Ronny Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany aut Schwaneberg Ulrich DWI-Leibniz Institut für Interaktive Materialien, Forckenbeckstraße 50, 52056, Aachen, Germany aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/12(2015-06-01), 5237-5246 0175-7598 99:12<5237 2015 99 253 https://doi.org/10.1007/s00253-015-6612-3 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6612-3 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Lülsdorf Nina DWI-Leibniz Institut für Interaktive Materialien, Forckenbeckstraße 50, 52056, Aachen, Germany aut NATIONALLICENCE 700 1- Vojcic Ljubica Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany aut NATIONALLICENCE 700 1- Hellmuth Hendrik International Research Laundry and Home Care, Biotechnology, Henkel AG & Co. KGaA, 40191, Düsseldorf, Germany aut NATIONALLICENCE 700 1- Weber Thomas International Research Laundry and Home Care, Biotechnology, Henkel AG & Co. KGaA, 40191, Düsseldorf, Germany aut NATIONALLICENCE 700 1- Mußmann Nina International Research Laundry and Home Care, Biotechnology, Henkel AG & Co. KGaA, 40191, Düsseldorf, Germany aut NATIONALLICENCE 700 1- Martinez Ronny Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany aut NATIONALLICENCE 700 1- Schwaneberg Ulrich DWI-Leibniz Institut für Interaktive Materialien, Forckenbeckstraße 50, 52056, Aachen, Germany aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/12(2015-06-01), 5237-5246 0175-7598 99:12<5237 2015 99 253