Many enzymatic activity assays are based on either (1) identifying and quantifying the enzyme with methods such as western blot or enzyme-linked substrate assay (ELISA) or (2) quantifying the enzymatic reaction by monitoring the changing levels of either product or substrate. We have generated an outer membrane protein G (OmpG)-based nanopore approach to distinguish enzyme identity as well as analyze the enzyme's catalytic activity. Here, we engineered an OmpG nanopore with a peptide cut site inserted into one of its loops to detect proteolytic behavior. In addition, we generated an OmpG nanopore with a single-stranded DNA attached to a loop for analyzing nucleolytic cleavage. This OmpG nanopore approach may be highly useful in analyzing specific enzymes in complex biological samples, or in directly determining kinetics of enzyme-substrate complex association and dissociation.
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