Chitosanase activity and relative purity assessment and assay of protease levels. (A) chitosanase activity; (B) protease activity; (C) SDS-PAGE of proteins in culture supernatants. The upper-table aligns the genotype of each strain and lists the type of medium for the corresponding columns in graphs (A) and (B) and wells of (C). Genetic symbols as in Fig. 4. Multi: chitosanase genes introduced on a multi-copy vector; Int: chitosanase genes introduced on an integrative vector. Culture media: Me: malt extract medium; Ch: chitosan flakes medium; Ol: medium with GlcN and chitosan oligomers. All determinations have been done after 72 h of culture. Data and error bars (A and B) are the mean of culture duplicates. *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05 from one-way ANOVA with Bonferroni's post test (GraphPad Prism version 5.00). (C) 20 μl of culture supernatants were loaded on a 12% SDS-PAGE gel. PageRuler™prestained protein ladder (0.5 μl; Fermentas) was used as standard. After electrophoresis, proteins were stained with Coomassie brilliant blue. Chitosanase migrates as a 26.5 kDa band.