Applied Microbiology and Biotechnology

, Volume 99, Issue 12, pp 5237–5246 | Cite as

A first continuous 4-aminoantipyrine (4-AAP)-based screening system for directed esterase evolution

  • Nina Lülsdorf
  • Ljubica Vojcic
  • Hendrik Hellmuth
  • Thomas T. Weber
  • Nina Mußmann
  • Ronny Martinez
  • Ulrich Schwaneberg
Methods and protocols


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 509 nm. The cBLE-4AAP screening system was optimized in 96-well microtiter plate format in respect to standard deviation (5 %), linear detection range (15 to 250 μM), lower detection limit (15 μM), 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 1036 U/mg and T3 929 U/mg) and shows compared to WT a 4.7-fold improved residual activity after thermal treatment (30 min incubation at 69.4 °C; WT 170 U/mg to T3 804 U/mg).


Directed evolution High-throughput screening Microtiter plate Esterase Thermal resistance 

Supplementary material

253_2015_6612_MOESM1_ESM.pdf (503 kb)
ESM 1(PDF 502 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Nina Lülsdorf
    • 1
  • Ljubica Vojcic
    • 2
  • Hendrik Hellmuth
    • 3
  • Thomas T. Weber
    • 3
  • Nina Mußmann
    • 3
  • Ronny Martinez
    • 2
    • 4
  • Ulrich Schwaneberg
    • 1
    • 2
  1. 1.DWI-Leibniz Institut für Interaktive MaterialienAachenGermany
  2. 2.Lehrstuhl für BiotechnologieRWTH Aachen UniversityAachenGermany
  3. 3.International Research Laundry and Home CareBiotechnology, Henkel AG & Co. KGaADüsseldorfGermany
  4. 4.EW Nutrition GmbH, Enzyme TechnologyCologneGermany

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