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Fast-track detection of novel enzymes
A high-throughput method for isolating proteases with high activity from a library of variants has been developed. As bait for a serine protease displayed on the surface of Escherichia coli, they fashioned a substrate consisting of a fluorescent dye adjacent to a positively charged moiety, a peptide bond that active proteases would cleave, and a quenching molecule that absorbs most of the dye s fluorescence energy. Following substrate cleavage by the protease, the negatively charged bacterial cell wall binds electrostatically to the positively charged fluorophore-containing product as the quencher-containing product is released. As a result, the cells fluoresce, allowing them to be quantified and isolated by fluorescence-activated cell sorting (FACS). The team screened a library of variants obtained by random mutagenesis for enzymes more active than the wild-type protease and found mutants that were up to 60 times more active. The ability to screen large libraries quantitatively by FACS may open new avenues to directed evolution of enzymes, they note
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