Fig. 5.
Detection of the 53D1-encoded chitinolytic protein from E. coli BL21 StarTM(DE3)/pET24b(+)::53D1 cells. a SDS-PAGE analysis of chromatography fractions. C.E.: crude extract; 1: flow-through; 2 and 3: fractions eluted at 125 and 500 mM imidazole, respectively. Std: standard reference protein, d-amino acid oxidase from Rhodotorula gracilis (42.5 kDa, 10 μg) gently provided by Loredano Pollegioni, University of Insubria, Italy. MW: molecular weight markers, from GE Healthcare Sciences, Little Chalfont, UK. 53D1 protein spot indicated by arrow. b Zymogram analysis in semi-native conditions of the purified 53D1 with carboxymethyl-chitin-remazol brilliant violet as substrate. 53D1 protein spot, visualized as a chitin degradation halo, indicated by arrow. Effect of pH (c), temperature (d), and increasing concentrations of NaCl (e) on chitobiosidase activity of the purified 53D1 protein, using 4-MU-(GlcNAc)2 as the substrate. Enzyme activities are reported as relative to the activity of 45.2 U/mg protein (set as 100 %) measured at pH 5, 37 °C and in the absence of salt. Values represent the means of three independent experiments (mean ± standard error)