Purification and characterization of a thermostable glucoamylase produced by Aspergillus flavus HBF34

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 Mn2+, Ca2+, Co2+ and Ba2+, it was inhibited by Hg2+, Fe3+, Al3+, Zn2+ and Cu2+. 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.