Purification and Characterization of Glucoamylase from Aspergillus niger ATCC 10864.
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Abstract
The Aspergillus niger ATCC 10864 produced substantial amount of glucoamylase activity when grown on rice bran as carbon source. The glucoamylase was purified using a procedure that included 80% saturated ammonium sulface precipitation dialysis, chromatography on Sephacryl S-100 and DEAE-Sepharose. Two fractions of glucoamylase from DEAE-Sepharose (F1a and F2a) were obtained. The purification fold were 26.24 and 19.57 times with 28 and 14.56 percent yields, the specific activities of 842.20 and 628.37 U/mg, and the molecular weight with estimated molecular mass of 80kDa and 73 kda, respectively. The optimal pH and temperature of F1a and F2a were 4.0, 60°C and 5.5, 60°C, respectively. F1 was stable at a pH range of 4.0 to 5.5 and at a temperature range of 20 to 50°C, while F2a was stable at a pH range of 4 to 6 and a temperature range of 20 to 40°C. In the presence of rice starch in fresh culture medium, the purified fractions of glucoamylase, F1a and F2a demonstrated apparent Km and Vmax values of 10 mg/ml, 0.02 umol/ml/min and 5.6 mg/mL, 0.032 umol/ml/min, respectively, while in the presence of soluble starch, the values of apparent Km and Vmax of F1 and F2 were 6.7 mg/ml, 0.02 umol/ml/min and 5.0 mg/ml 0.023 umol/ml/min, respectively.
Keywords: glucoamylase, purification, characterization, Km, Vmax
Corresponding Author Email: kkaree@kmitl.ac.th
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