Fig 3. Enzymatic activity of chitinases ChiA and ChiB towards (A) pseudo-chitin substrates (effect temperature and pH) or (B) crude chicken mucus.

(A) Effect of temperature and pH on chitinase activity towards pseudo-chitin substrates (4-MU-GlcNAc₂ and 4-MU-GlcNAc₃). The enzymatic activity of ChiA (left) or ChiB (right) at different temperatures was assessed at a constant pH of 5 (upper panels), whereas the effect of the pH was monitored at a constant temperature of 42°C (lower panels) (no replicates). (B) Chitinase activity towards chicken mucus using turbidity assay. Biological replicates of crude intestinal chicken mucus (150 μg) were incubated with either recombinant ChiA, ChiB (15 μg) or PBS. After a one hour incubation period at 37°C, the turbidity of the mixture was measured at an OD-value of 450 nm. The relative index was calculated by dividing the OD of the chitinase-treated mucus by the OD of the untreated mucus sample for each mucus sample. Lines indicate the means with their respective standard deviations. Significant differences are indicated with ‘*’ (p≤0.05). ‘**’ (p≤0.01) and ‘***’ (p≤0.001).