Isolation, purification and characterization of glucoamylase from Aspergillus sp. 3104 and isolate 1

Glucoamylase was produced from Aspergillus sp. BIOTECH 3104 and bacterial Isolate 1 (ISO 1) by solid substrate cultivation and shake flask method, respectively. The soluble glucoamylase was separated from biomass by centrifugation. The culture supernatant of Aspergillus sp BIOTECH 3104 underwent ultrafiltration before the enzyme was precipitated by 90% amomnium sulfate while the culture supernatant of ISO was immediately precipitated by 90% ammonium sulfate. Glucoamylase from isolates BIOTECH 3104 and ISO 1 were desalted by dialysis and gel filtration chromatography using Sephadex G-25. Dialysates were further purified through ion exchange chromatography using DEAE-cellulose and gel filtration chromatography using Sephadex G-100. The enzymes were assayed for volumetric activity, protein content and specific activity at each step of purification. The molecular weights of the purified glucoamylases were determined by SDS-PAGE [sodium dodecyl sulfate-poly-acrylamide gel electrophoresis]. The effects of pH, temperature, inhibitors (1 mM EDTA and metal ions) and denaturing agents (2% SDS and 0.8 M urea) on the volumetric activity of the purified glucoamylases were determined. The effect of storage time on shelf life of the glucoamylases was determined for four consecutive weeks using 1% sodium benzoate and 1% potassium sorbate as preservatives. Results were statistically evaluated using simple completely randomized design. Analysis of variance (ANOVA) was used to determine the significant differences between means. Results revealed that glucoamylase purified from Aspergillus sp. BIOTECH 3104 had a molecular weight of ~60 kDa. This enzyme was stable at pH 4-5 and at 30 deg C to 50 deg C. The most potent inhibitors of the fungal glucoamylase were EDTA, Fe3+ and CO2+. The volumetric activity was reduced to less than 40% by 1% SDS and 0.8 M urea. The use of 1% sodium benzoate as preservative retained 91.92% glucoamylase activity whereas the use of 1% potassium sorbate as preservative retained 86.16% glucoamylase activity adter four weeks of storage. Glucoamylase purified from the bacterial isolate ISO 1 showed ~60 kDa molecular weight. It was optimally active at pH 7-8 and manifested a very narrow thermal stability (40 deg C). The enzyme was readily inactivated by EDTA and Fe3+. Addition of 1% SDS and 0.8 M urea completely denatured the bacterial glucoamylase. The use of 1% sodium benzoate as preservative retained 53.13% glucoamylase activity whereas the use of 1% potassium sorbate as preservative retained 37.06% glucoamylase activity after four weeks of storage