A raw starch degrading a-amylase from Bacillus sp. WN11 was
used to hydrolyse the cassava starch. The Bacillus sp. WN11 cells
were cultivated in a medium defined by Mamo and Gessesse
(1997). A 100 mL sterile medium in a 250 mL shake flask was inoculated
with Bacillus sp. WN11 cells and incubated for 24 h at 55 C
and 180 rpm shaking. This culture was used to inoculate (10%, v/v)
the 250 mL medium (with same composition) in 1 L shake flask.
The inoculated production culture was cultivated in the same
way the seed culture has been grown. The culture was subsequently
centrifuged at 4 C and 5500g and the clear supernatant
was filter concentrated (17-fold) using a 10 kDa cut-off membrane
(Pellicon 2, Biomaxpolyethersulfone, Sterlitech Corporation, USA).
The concentrated enzyme was equilibrated with 50 mM Tris–HCl
buffer, pH 7.0 and stored at 4 C until used to hydrolyse the cassava
starch. About 1.8 mL (19.2 U) of the enzyme preparation was used
to hydrolyse 17 g/L of soluble starch in 20 mM sodium acetate buffer,
pH 5.5, and incubated at 60 C for 10 min. One unit of the
enzyme activity is defined as the amount of enzyme that releases
1 lmol of reducing sugar per minute under the assay conditions.
2.3