**3.6. Effect of temperature and pH on activity and stability**

The optimal temperature for amylase activity was determined by assaying activity between 30 and 100°C for 30 min in 50 mM phosphate buffer. Measurement of optimum pH for amylase activity was carried out under the assay conditions for pH range of 3.0-10.0, using 50mM of three buffer solutions: Tris-HCl (pH 3.0), Na2HPO4-Citrate (pH 4.0 – 6.0), and Glycine-NaOH (pH 7.0-10.0).

The temperature stability was determined by incubating the partial purified enzyme solution in water bath for temperature range of 30-100°C for 30, 60, 90, 120, 180 min and then cooled with tap water. The remaining -amylase activity was measured and expressed as the percentage of the activity of untreated control taken as 100%. The first order inactivation rate constants, ki were calculated from the equation: lnA lnA k t 0 i , where A0 is the initial value of amylase activity and A the value of amylase activity after a time t (min).

For the determination of pH stability, the enzyme was incubated in a water bath at 60 °C at varying pH value for 30 min. The residual activity was detected under the same conditions and expressed as the percentage of the activity of untreated control taken as 100%.

### **3.7. Effect of metal salts and chelating agent**

The effect of metal salts and EDTA on amylase activity was determined by adding 0.05 to 0.1% (w/v) of metal salts (CaCl2.2H2O, MgSO4.7H2O, FeSO4.7H2O, NaCl, FeCl3, CuSO4.5H2O) and EDTA to the standard assay. The effect of metal salts and chelating agent on amylase activity were evaluated by pre-incubating the enzyme in the presence of effectors for 30 min at 60°C. The remaining amylase activity was determined and expressed as the percentage of the activity of untreated control taken as 100%.
