EFFECT OF CULTURAL CONDITIONS ON THE PRODUCTION OF α-AMYLASE FROM FERMENTED COCOA BEANS USING SUBMERGE FERMENTATION TECHNIQUES

This study identifies the best cultural conditions and nutritional requirements for the selected fungi species isolated from cocoa beans for production of α-amylase and this was studied in 250ml Erlenmeyer flasks. Six different fermentation media (M1–M6) of different compositions were evaluated for this purpose. The medium containing M5 medium (g/l); starch 20, yeast extracted 8.5 NH4Cl 1.3, MgSO4.7H2O 0.12, CaCl2 0.06 gave maximum enzyme production. Effect of inoculums size, incubation period, temperature, pH, metal ions, and chemical compounds were investigated on the enzyme production and was observed that these conditions can affect the rate of enzyme production. However, Optimum enzyme production of the fungi (Aspegillus oryza) was obtained at 72 hours of incubation at temperature of 30ºC, pH 5, inoculum size 4% and optimum level of ammonium sulphate precipitated produced saturation level of 70% that gave 1.3 fold enzyme purification. Using Sephadex-DEAE column, the active fractions were eluted using 0.05 M Tris-HCl buffer containing 0.30 M NaCl at pH 7.5. The molecular weight of α-amylase was found to be 47 kDa on SDS-PAGE after gel filtration. The maximum activity was achieved after 30 min at 40ºC and pH 5 in the presence of Ca+2 ion.