A first continuous 4-aminoantipyrine (4-AAP)-based screening system for directed esterase evolution
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| 008 | 210128e20150601xx s 000 0 eng | ||
| 024 | 7 | 0 | |a 10.1007/s00253-015-6612-3 |2 doi |
| 035 | |a (NATIONALLICENCE)springer-10.1007/s00253-015-6612-3 | ||
| 245 | 0 | 2 | |a A first continuous 4-aminoantipyrine (4-AAP)-based screening system for directed esterase evolution |h [Elektronische Daten] |c [Nina Lülsdorf, Ljubica Vojcic, Hendrik Hellmuth, Thomas Weber, Nina Mußmann, Ronny Martinez, Ulrich Schwaneberg] |
| 520 | 3 | |a 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). | |
| 540 | |a Springer-Verlag Berlin Heidelberg, 2015 | ||
| 690 | 7 | |a Directed evolution |2 nationallicence | |
| 690 | 7 | |a High-throughput screening |2 nationallicence | |
| 690 | 7 | |a Microtiter plate |2 nationallicence | |
| 690 | 7 | |a Esterase |2 nationallicence | |
| 690 | 7 | |a Thermal resistance |2 nationallicence | |
| 700 | 1 | |a Lülsdorf |D Nina |u DWI-Leibniz Institut für Interaktive Materialien, Forckenbeckstraße 50, 52056, Aachen, Germany |4 aut | |
| 700 | 1 | |a Vojcic |D Ljubica |u Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany |4 aut | |
| 700 | 1 | |a Hellmuth |D Hendrik |u International Research Laundry and Home Care, Biotechnology, Henkel AG & Co. KGaA, 40191, Düsseldorf, Germany |4 aut | |
| 700 | 1 | |a Weber |D Thomas |u International Research Laundry and Home Care, Biotechnology, Henkel AG & Co. KGaA, 40191, Düsseldorf, Germany |4 aut | |
| 700 | 1 | |a Mußmann |D Nina |u International Research Laundry and Home Care, Biotechnology, Henkel AG & Co. KGaA, 40191, Düsseldorf, Germany |4 aut | |
| 700 | 1 | |a Martinez |D Ronny |u Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany |4 aut | |
| 700 | 1 | |a Schwaneberg |D Ulrich |u DWI-Leibniz Institut für Interaktive Materialien, Forckenbeckstraße 50, 52056, Aachen, Germany |4 aut | |
| 773 | 0 | |t Applied Microbiology and Biotechnology |d Springer Berlin Heidelberg |g 99/12(2015-06-01), 5237-5246 |x 0175-7598 |q 99:12<5237 |1 2015 |2 99 |o 253 | |
| 856 | 4 | 0 | |u https://doi.org/10.1007/s00253-015-6612-3 |q text/html |z Onlinezugriff via DOI |
| 898 | |a BK010053 |b XK010053 |c XK010000 | ||
| 900 | 7 | |a Metadata rights reserved |b Springer special CC-BY-NC licence |2 nationallicence | |
| 908 | |D 1 |a research-article |2 jats | ||
| 949 | |B NATIONALLICENCE |F NATIONALLICENCE |b NL-springer | ||
| 950 | |B NATIONALLICENCE |P 856 |E 40 |u https://doi.org/10.1007/s00253-015-6612-3 |q text/html |z Onlinezugriff via DOI | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Lülsdorf |D Nina |u DWI-Leibniz Institut für Interaktive Materialien, Forckenbeckstraße 50, 52056, Aachen, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Vojcic |D Ljubica |u Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Hellmuth |D Hendrik |u International Research Laundry and Home Care, Biotechnology, Henkel AG & Co. KGaA, 40191, Düsseldorf, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Weber |D Thomas |u International Research Laundry and Home Care, Biotechnology, Henkel AG & Co. KGaA, 40191, Düsseldorf, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Mußmann |D Nina |u International Research Laundry and Home Care, Biotechnology, Henkel AG & Co. KGaA, 40191, Düsseldorf, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Martinez |D Ronny |u Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Schwaneberg |D Ulrich |u DWI-Leibniz Institut für Interaktive Materialien, Forckenbeckstraße 50, 52056, Aachen, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 773 |E 0- |t Applied Microbiology and Biotechnology |d Springer Berlin Heidelberg |g 99/12(2015-06-01), 5237-5246 |x 0175-7598 |q 99:12<5237 |1 2015 |2 99 |o 253 | ||