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

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).

Inhibition of the D-alanine:D-alanyl carrier protein ligase from Bacillus subtilis increases the bacterium's susceptibility to antibiotics that target the cell wall.

The surface charge as well as the electrochemical properties and ligand binding abilities of the Gram-positive cell wall is controlled by the D-alanylation of the lipoteichoic acid. The incorporation of D-Ala into lipoteichoic acid requires the D-alanine:D-alanyl carrier protein ligase (DltA) and the carrier protein (DltC). We have heterologously expressed, purified, and assayed the substrate selectivity of the recombinant proteins DltA with its substrate DltC. We found that apo-DltC is recognized by both endogenous 4'-phosphopantetheinyl transferases AcpS and Sfp. After the biochemical characterization of DltA and DltC, we designed an inhibitor (D-alanylacyl-sulfamoyl-adenosine), which is able to block the D-Ala adenylation by DltA at a K(i) value of 232 nM vitro. We also performed in vivo studies and determined a significant inhibition of growth for different Bacillus subtilis strains when the inhibitor is used in combination with vancomycin.