One gram of the culture of SSF from each flask
was mixed with 8 ml of deionized water and acidwashed
sand in an ice-cold mortar. The homogenate
was centrifuged at 2000g for 5 min. The supernatant
was assayed for a-amylase activity within 12 h of
preparation. Amylase (a-1, 4-glucan-4-glucanohydrolase,
EC 3.2.1.1) was assayed with the substrate
1% soluble starch in 20 mM phosphate buffer (pH
6.9). A mixture of 0.5 ml of extract and 0.5 ml of 20
mM phosphate buffer (pH 6.9) containing 1% soluble
starch was incubated at 25jC for 3 min. The
reducing sugars released therein were then determined
by the method of Bernfeld (1955) with
reference to a standard curve of maltose. One aamylase
unit (U) was defined as the amount of
enzyme producing 1 Amol of maltose per min at
25jC on soluble starch.