Figure 5
Detection of α-amylase anchored on the cell wall of four different strains using either the YhcR123 sorting sequence with the 123-aa spacer by Western blot. All B. subtilis strains were inoculated to an OD578 of 0.05 - 0.08 in LB medium. After 1 h of growth, 0.1 mM IPTG was added to induce expression of the hybrid amyQ gene and cells were collected 4, 8 and 12 h after further inoculation. Equal amounts of cells were treated with lysozyme to release the anchored α-amylase. The samples were applied to SDS-PAGE and Western blot as described (Nguyen and Schumann 2006). Strains NDH31 (ΔywpE, ΔyhcS; lane 1), NDH30 (ywpE+, ΔyhcS, lane 2) SZ60 (ΔywpE, yhcS+; lane 3) and 1012 (ywpE+, yhcS+; lane 4), all of them carrying the plasmid pNDH89 (AmyQ-YhcR123). HtpG, a cytoplasmic protein, was used as loading control for the proteins released from the cytoplasm.