2.4. Glucoamylase activity assay and kinetic analysis

Glucoamylase activity was measured by determining free glucose released after maltose hydrolysis. The reaction mixture consisted of 0.1 M sodium phosphate buffer (pH 6.0) containing 2% (w/v) maltose, 0.032 μg/μl of SdGA at final volume 100 μL at 39 °C for 20 min. The amount of released glucose was determined using a commercial kit based on the method of glucose oxidase (GOD)/peroxidase (POD) (Wiener laboratories, Rosario, SF, Argentina) [32]. One unit of GA hydrolytic activity was defined as the amount of enzyme needed to release 1 μmol of glucose per min. at 39 °C. Assays were performed at least by triplicate at different enzyme concentrations to ensure steady-state conditions. Protein concentrations were estimated according to the method of Bradford [33].

The effect of maltose concentration on SdGA activity was evaluated in 0.1 M sodium phosphate buffer (pH 6.0) at 39 °C. The kinetic parameters K_m, k_cat, S_0.5, V_max and n_H were calculated with a computer program using the Levenberg-Marquardt algorithm for regression by fitting the data to the Hill equation [34]. S_0.5 is defined as the concentrations of substrate that give 50 % maximal activity (V_max), k_cat is the turnover number and n_H is the Hill number.

The effect of pH on the activity of SdGA was determined in two 0.1 M buffers with maltose as the enzyme substrate (sodium acetate buffer, pH 4–5.6 and sodium phosphate buffer, pH 6–8.0, respectively) at 39 °C. The effect of temperature on SdGA activity was determined with 2% (w/v) maltose between 10–50 °C as described above. Temperature stability assays were performed by incubating the same quantity of SdGA at different temperatures for 0–30 min., and the residual activity was measured under standard assay conditions. The effect of metabolites on the thermal stability of SdGA was measured using 0.1 mM acarbose (Sigma-Aldrich cat. n° 56180-94-0), 1 mM CaCl₂, 10 % (v/v) glycerol or 1 M NaCl. The first order equation ln (A/A₀) = -k. t was used to calculate the inactivation rate constant (k, min⁻¹). A is the residual activity at time t and A₀ is the initial activity at time zero. D-value (Decimal reduction time), the time to reduce the initial activity 90 %, was calculated from the equation: D = ln(10) / k as described [35]. All measurements were made at least by triplicate. Significant differences were determined by one-way ANOVA and Dunnet test using GraphPad Prism version 5.0 software (GraphPad Software, La Jolla, CA, USA)