FIGURE 5.

Expression of NukT mutants in S. carnosus and their peptidase activity in vitro. ISO vesicles were prepared from cells of recombinant S. carnosus, and 5 μg of membrane protein was subjected to SDS-PAGE, followed by Coomassie Brilliant Blue staining (A). The arrow indicates the position of NukT (80 kDa). Modified His-NukA (3 μm) was incubated with 1 mg/ml NukT membrane. Nukacin ISK-1 produced after the reaction was detected from its antibacterial activity by using the overlay assay method with L. sakei as the indicator strain (B), and the remaining substrates in the reaction mixture were visualized using Western blotting with anti-His tag antibody (C).