PRODUCTION OF Aspergillus niger GLUCOAMYLASE USING GUINEA CORN STARCH AMYLOPECTIN AS THE ONLY CARBON SOURCE
ABSTRACT
This study was aimed at the production of glucoamylase which can be utilised for starch hydrolysis. A fourteen days experimental study was carried out to determine the day of highest glucoamylase activity. Day five and day twelve of the fourteen days experimental study had the highest glucoamylase activity. The specific activity for the crude enzyme was found to be 729.45 U/mg for glucoamylase isolated from Aspergillus niger in submerged fermentation using amylopectin fractionated from guinea corn starch as the carbon source after five days of fermentation (GluAgGC5), and 1046.82 U/mg for glucoamylase isolated from Aspergillus niger in submerged fermentation using amylopectin fractionated from guinea corn starch as the carbon source after twelve days of fermentation (GluAgGC12).The crude enzyme was purified by ammonium sulphate precipitation and by gel filtration (using sephadex G 100 gel). Ammonium sulphate saturations of 70% and 20% were found suitable to precipitate proteins with highest glucoamylase activity. After ammonium sulphate precipitation, the specific activities of the enzyme were found to be 65.98 U/mg and 61.51 U/mg for GluAgGC5 and GluAgGC12, respectively. Similarly, after gel filtration, the specific activities of the enzyme were found to be 180.52 U/mg and 272.81 U/mg for GluAgGC5 and GluAgGC12, respectively. The optimum pH for GluAgGC5 were found to be 7.5,7.5 and 6.0 when using tiger nut starch, cassava starch and guinea corn starch as substrates, respectively, while the optimum pH for GluAgGC12 were found to be 5.0, 8.5 and 7.0 when using tiger nut starch, cassava starch and guinea corn starch as substrates, respectively. The enzyme activity in GluAgGC5 was enhanced by Ca2+,Co2+, Fe2+, Mn2+and Zn2+ but Pb2+ had inhibitory effect on the enzyme. Similarly, the enzyme activity of GluAgGC12 was enhanced by Ca2+, Zn2+, Co2+, Fe2+ and Mn2+ while Pb2+ had inhibitory effect on the enzyme. The optimum temperatures were found to be 50˚C and 45˚C for rGluAgGC5 and GluAgGC12, respectively. The Michaelis Menten’s constant, Km and maximum velocity Vmax of GluAgGC5 obtained from the Lineweaver-Burk plot of initial velocity data at different substrate concentrations were found to be 770.75 mg/ml and 2500µmol/min using cassava starch as substrate, 158.55 mg/ml and 500 µmol/min using guinea corn starch as substrate and 46.23 mg/ml and 454.53µmol/min using tiger nut starch as substrate. Also, the Km and Vmax of GluAgGC12 were found to be 87.1 mg/ml and 384.61µmol/min using cassava starch as substrate, 29.51 mg/ml and 243.90 µmol/min using guinea corn starch as substrate and 2364 mg/ml and 2500µmol/min using tiger nut starches as substrate.