Fig. 4.

Effect of temperature (A), sodium chloride concentration (B), pH (C) and addition of chelators or cations on the activity of AlyA3. (A–D) Experiments were conducted with sodium alginate 0.22% as substrate and 6 nM of the purified enzyme. Activity was monitored by following the increase in absorbance at 235 nm during the first 2 min. Error bars indicate standard deviations of three experimental replicates. (A) Effect of temperature. Experiments were conducted in 0.1 M Tris–HCl buffer, pH 7.5. Activity at 40°C was taken as 100%. (B) Effect of NaCl concentration. Reactions were conducted in 0.1 M Tris–HCl buffer, pH 7.0 at 25°C. Activity with 2 mM NaCl was taken as 100%. (C) Effect of pH on AlyA3 activity. Experiments were performed at 25°C in 100-m buffer. Sodium citrate (stripes), MES (white), Tris–HCl (dark gray), HEPES (light gray) and CHES (black) buffers were used in the assay. Activity in Tris–HCl, pH 7.0, was taken as 100%. (D) Effect of chelators and cation chloride addition on AlyA3 activity. Reactions were conducted in 0.1 M Tris–HCl buffer, pH 7.0 at 25°C. Either CaCl₂, LiCl₂, MgCl₂ (2 mM), EDTA (2 or 10 mM) or EGTA (2 or 10 mM) were added. Activity without any cation chloride or chelator was taken as 100%. This figure is available in black and white in print and in color at Glycobiology online.