Griswold Group: High Throughput Ester Hydrolase Screens

 

Although not active at this time, we are preparing to initiate studies into novel approaches to ultra-high throughput functional screening of esterases, lipases and alkyl sulfatases. Fluorescence techniques are the key to quantitative, real-time assessment of enzymatic hydrolysis. New detection methods will be combined with cutting edge microbial display platforms, and ultimately the projects will be steered towards design and isolation of enzymes with practical utility in the synthesis of pharmaceuticals and other organic compounds.

Figure 1 - Microcompartmentalization of E. coli expressing highly active recombinant esterase in top row and inactivated mutant form of the enzyme in bottom row. Left Panel: phase contrast showing 10-40 um compartments consisting of water-in-oil-in-water double emulsions. Middle Panel: fluorescence image of same field of view using filter for blue fluorescence. Recombinant E. coli expressing BFP are seen in top panel (two cells) and bottom panel (one cell). Bacteria reside in internal aqueous microdroplets of compartments. Image brightness enhanced to highlight bacterial cells. Right Panel: fluorescence image of same field of view using filter for green fluorescence. Recombinant E. coli expressing active esterase in top image hydrolyze the non-fluorescent fluorescein dibutyrate substrate liberating the fluorescein fluorophore and resulting in bright green fluorescence of the respective inner aqueous microdroplet. Recombinant E. coli expressing inactive variant enzyme in bottom image are unable to hydrolyze the non-fluorescent substrate, and do not generate green fluorescence in their aqueous microdroplets. Far Right Panel: photograph of bulk suspension of microdroplets reveals strong green fluorescence of compartments containing active cells and absence of green fluorescence in compartments containing inactive cells.

 

Figure 2 - Results of mock library screening experiments. E. coli cells expressing wild type or inactive forms of the esterase enzyme were mixed at specified ratios, compartmentalized with fluorescein dibutyrate as above, and the compartments were sorted on a flow cytometer isolating compartments showing a high level of green fluorescence. The bar graph indicates the percentage of a population represented by wild type enzyme expressing cells both before (blue) and after (red) sorting. The enrichment factor ([final% WT]/[initial% WT]) is indicated by the white bars on the secondary axis. Two different display formats were tested. On the left is soluble periplasmic expression of the enzymes, and on the right is surface anchored expression of the enzymes.