After electrophoresis, gel was stained with Coomassie Brilliant Blue R-250. For activity staining, zymographic analysis of the protease was performed using gelatin (0.1%) as the substrate as Raf inhibition described by Karbalaei-Heidari et al. (2009). Zymographic analysis for the amylase was performed on nondenaturing electrophoresis slab gels (10% polyacrylamide) prepared with 10% of sucrose, as described by Cadenas & Engel (1994). The amylase activity, with soluble starch as the substrate,

was determined using DNS (3,5-dinitrosalicylic acid) method (Miller, 1959). One unit (U) of amylase activity was defined as the amount of enzyme necessary to produce 1 μmol of reducing sugar per minute under the assay conditions. Protease activity was measured as described previously (Karbalaei-Heidari et al., 2009). One unit (U) of protease activity was defined as the amount of enzyme yielding 1 μmol of tyrosine per minute under the assay conditions. The effect of pH on enzyme activity was studied over a pH range of 4.0–12.0. The pH stability of the enzymes was determined by incubation with different buffer systems at 30 °C for 24 h. The following buffer systems (100 mM) were used: glycine-HCl buffer, pH 4.0; sodium acetate buffer, pH 5.0–6.0; potassium phosphate buffer, pH 7.0; Tris–HCl buffer, pH 8.0–8.5; glycine–NaOH buffer, pH 9.0–12.0. To investigate the effect of temperature, the assay

was conducted under different temperatures from 30 to 90 °C. The thermostability of the enzyme was determined by pre-incubating Enzalutamide solubility dmso the enzyme sample at various temperatures for 24 h, and residual activity was measured Florfenicol using the standard assay. The activity of the purified enzyme was measured in enzyme reaction mixture containing 0–20% NaCl. Salt stability of the enzyme was determined by incubating

the enzyme with different concentrations of NaCl for 24 h, and the remaining activity was determined under standard assay conditions. The effect of organic solvents with different log Pow values at 50% (v/v) concentration on the purified enzyme was determined by incubating the enzyme solution in different organic solvents at 30 °C. Residual activity was measured under the standard conditions. If residual activity was more than 50% after 10 days, half-life was taken as ‘> 10 days’. While activity was < 50% after 1 h, half-life was taken as ‘< 1 h’. Effects of different metal ions and chemical reagents [ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), phenylarsine oxide (PAO), diethyl pyrocarbonate (DEPC), β-mercaptoethanol, SDS, Triton X-100, and Tween-80] on the activity of purified enzymes were examined after they had been pre-incubated with them at 30 °C for 12 h, respectively, and the residual activity was determined under optimal assay conditions. Activity of the enzyme assayed in the absence of any additives was taken as 100%.