Figure 6

α-Amylase activities in the presence and absence of potential sortases. The following strains were analyzed: 1012/pNDH37 (basic expression vector with the IPTG-inducible promoter Pgrac and the signal sequence of amyQ), 1012/pNDH37-amyQ (secretes α-amylase into the medium), NDH03/pNDH19 (contains the xylose-inducible srtA of L. monocytogenes and amyQ fused to the sorting sequence of FnBPB), NDH30 (ΔyhcS)/pNDH89 (AmyQ-YhcR123) and 1012/pNDH89. Cells were grown to the mid log-phase and then, 0.5 mM IPTG and 0.5% xylose were added into all five cultures to induce production of sortase A (strain NDH03), wild-type amylase (pNDH37-amyQ) and hybrid α-amylase (from pNDH19 and pNDH89). Samples were collected after 4 h of induction and the cells were separated from the growth medium by centrifugation. α-Amylase activities were determined with whole cells that the number of cells are identical in all probes and with the supernatant from strain 1012/pNDH37-amyQ. The activities were presented as relative activity (%), where the activity measured with the supernatant from 1012/pNDH37-amyQ was set at 100%.