Abstract:
Glucoamylase (GA) from Aspergillus flavus HBF34 strain was partially purified 120 folds using starch
affinity chromatography. Two isoenzymes (GA1 and GA2) were identified by polyacrylamide gel
electrophoresis (PAGE) zymography. Sodium dodecyl sulfate (SDS)-PAGE analysis revealed that one
of the enzymes consist of one subunit and the other, two subunits. The optimum pH of the purified GA
was 6.0 and the optimum temperature was 60° C. GA was found to be stable at temperatures up to 50° C
and at a pH range between 3.0 and 9.0. Km and Vmax values of the enzymes were determined using
soluble potato starch, glycogen, amylopectin and amylose as substrates and calculated to be 0.046,
0.075, 0.1 and 0.125 mg/ml and 769, 1250, 3333 and 2500 U/mg protein, respectively. While GA was
activated by Mn
2+
, Ca
2+
, Co
2+
and Ba
2+
, it was inhibited by Hg
2+
, Fe
3+
, Al
3+
, Zn
2+
and Cu
2+
. The activity of
GA was found to be tolerant up to 5 M NaCl concentration. N-bromosuccinimide (NBS) and
phenylmethanesulfonylfluoride (PMSF) inhibited the enzyme, suggesting the involvement of
tryptophan and serine residues in the catalytic process. Raw corn starch adsorption of GA was found
to be 93%. Thin-layer chromatography (TLC) results showed that amylase was in fact a glucoamylase.
