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

Abstract

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).

Microbial enzymes for the recycling of recalcitrant petroleum‐based plastics: how far are we?

“…A number of lipases (Vertommen et al ., ; Eberl et al ., ; Ronkvist et al ., ), esterases (Liebminger et al ., ) and cutinases (Müller et al ., ; Alisch‐Mark et al ., ; Araujo et al ., ; Nimchua et al ., ; Ronkvist et al ., ; Herrero Acero et al ., ; Chen et al ., ; Wei et al ., ) from fungal and actinomycete species hydrolyse amorphous PET and modify the surface of PET films and fibres (Zimmermann and Billig, ). Carboxylesterases from Bacillus licheniformis , Bacillus subtilis and Thermobifida fusca also partially hydrolysed PET fibres and showed a high activity against PET oligomers (Billig et al ., ; Oeser et al ., ; Ribitsch et al ., ; Lülsdorf et al ., ; Barth et al ., ). Lipases display low activity against PET due to their lid structure covering the buried hydrophobic catalytic centre and the resulting limited accessibility for polymeric substrates (Guebitz and Cavaco‐Paulo, ; Eberl et al ., ; Zimmermann and Billig, ).…”

FoldX as Protein Engineering Tool: Better Than Random Based Approaches?

“…However, in case of enzymes, selection might easily be performed by heating up unpurified crude extracts from cells [ 42 ]. Using this technique, protein melting temperature T m can be improved in the best screenings by far >10 °C [ [42] , [43] , [44] , [45] , [46] , [47] ]. The artificial evolution approach can result in a 140-fold increase in long-term enzymatic activity like demonstrated for the alkaline pectate lyase [ 48 ].…”

A robust protocol for directed aryl sulfotransferase evolution toward the carbohydrate building block GlcNAc

“…The key performance parameter for a robust screening system in directed evolution is comprised of the coefficient of variation (CV) which should be below 15% (Lehmann et al, ; Lülsdorf et al, ; Tee & Schwaneberg, ). Optimized parameters are the employed cell lysate volume as well as donor and acceptor concentrations.…”

“…Stepwise optimization of screening parameters yielded finally a CV of 14.3%. MTP based screening systems with a true CV below 15% are routinely and successfully employed in directed evolution campaigns (e.g., cellulase, Lehmann et al, ; esterase, Lülsdorf et al, ; and epoxygenase, Tee et al, ). The p NPS screening system was validated in one round of directed evolution employing a random ASTB mutagenesis library, which yielded after screening of 1,760 clones ASTB variants with an up to 3.4‐fold improved GlcNAc sulfurylation activity.…”