After 10 min of incubation at ICC, the liberated reducing sugars (glucose equivalents) were estimated by the dimensionality’s acid (DNS) method of Miller (Miller et al. , 1959). The blank contained 0. 5 ml of 0. 1 M acetate buffer (pH 5. 0), 1. 25 ml of 1% starch solution and 0. 25 ml of distilled water. One unit (10) of amylase is defined as the amount of enzyme releasing 1 mol glucose equivalent per minute under the assay notations. Innocuous preparation 5 ml of medium containing nutrient broth was transferred to a 50 ml tube and sterilized in an autoclave at 121 co for 20 min.
After cooling, a loophole of bacterial culture was aseptically transferred and rotated at 200 RPM (ICC) in a shaking incubator overnight. 1% of this culture was used to inoculate 20 ml of the same medium in 100 ml flask and incubated in orbital shaker at ICC until the optical density at Mann (60TH) reached 0. 15 (cell density about xx colony-forming unit Screening of amylase producing bacteria 25 PL of the prepared innocuous room each of BacillUS isolates was aliquots onto starch agar plates contained (0. 1 % Removal Starch), incubated at ICC for 72 h and screened for amylase detection.
Isolates having a higher ratio of clearing zone to colony size were chosen for further investigation. Effect of medium pH and temperature incubation The influence of initial medium pH on amylase production was assessed by cultivating the strain in the basal media of pH ranging from 3. 0 to 11. 0 The effect of temperature was studied by performing the 52 Washer Baker et al. / Research in Biotechnology, 3(6):51-58, 2012 was added and photographed under UP eight.