Enzyme kinetic and specific activity assays.

Enzyme concentration was determined by Bio-Rad protein assay (Bio-Rad Laboratories). Enzyme activity was assayed by detecting the reducing sugars at OD₅₄₀ according to the DNS (3,5-dinitro-2-hydroxybenzoic acid) method (20). The enzyme specific activity was tested in 50 mM sodium-acetate buffer, pH 5.5, with 1% carboxymethylcellulose sodium salt (CMC; low viscosity; Sigma-Aldrich) as the enzyme substrate at 60°C for 5 min. One unit of activity is defined as the amount of enzyme releasing 1 μmol reducing equivalents per minute. Kinetic parameters (K_m and k_cat) were determined with the CMC concentration varied from 0.5 to 2.5% at 60°C for 3 min using a fixed enzyme concentration (1 μg 200 μl⁻¹). The activity data were fit to the Michaelis-Menten equation to determine K_m and k_cat by GraphPad Prism 5. Averages from triplicate experiments are displayed in Table 1.

Kinetic parameters and specific activity of wild-type AaCel9A and its mutants