Isolation, partial purification and characterization of glucoamylase from aspergillus niger in submerged fermentation using amylopectin from tiger nut starch as carbon source

Thesis Abstract

Glucoamylase was isolated from Aspergillus niger and purified using ammonium sulphate precipitation and gel filtration respectively. The purified enzyme was then characterized to determine the optimum conditions required by the produced enzyme. A fourteen day pilot study was carried out to determine the day of isolation of crude with highest glucoamylase activity. Day 5 and 12 had highest glucoamylase activity. The specific activities for the crude enzyme were found to be 757.5U/mg and 1223.88U/mg for glucoamylase isolated from Aspergillus niger in submerged fermentation using amylopectin fractionated from tiger nut starch as the carbon source after five days of fermentation (GluAgTN5) and twelve days of fermentation (GluAgTN12). Ammonium sulphate (20% and 90%) saturation was found suitable to precipitate protein with highest glucoamylase activity for GluAgTN5 and GluAgTN12, respectively. Following ammonium sulphate precipitation and gel filtration, the specific activities were found to be 89.90U/mg and 276.03U/mg for GluAgTN5, While for GluAgTN12, the specific activities were 88.75U/mg and 80.95U/mg following ammonium sulphate precipitation and gel filtration, respectively. The roptimum pH and temperature for GluAgTN5 were found to be 6.5, 7.0, 6.0 at 55°C and 8.5, 6.0, 7.5 at 50°C for GluAgTN12 using cassava, guinea corn and tiger nut starch as substrates. The enzyme activity in GluAgTN5 was enhanced by Ca2+ and Fe2+ while Zn2+ and Co2+ had inhibitory effects, Mn2+and Pb2+, however completely inactivated the enzyme. The enzyme activity in GluAgTN12 was enhanced by Ca2+ while Co2+and Zn2+ had inhibitory effects, Fe2+, Mn2+ and Pb2+ completely inactivated the enzyme. The Michealis-Menten constant, Km and maximum velocity, Vmax obtained from Line-Weaver-Burk plot of initial velocity data at different substrate concentrations were found to be 222mg/ml and 500µmol/min using cassava starch, 291mg/ml and 1000µmol/min using guinea corn starch, 137.5mg/ml and 500µmol/min using tiger nut starch as substrate for GluAgTN5. While for GluAgTN12, Km and Vmax were found to be 176.6mg/ml and 100µmol/min using cassava starch, 491mg/ml and 1000µmol/min using guinea corn starch, 131.5mg/ml and 500µmol/min using tiger nut starch as substrate.