PARTIAL PURIFICATION AND CHARACTERIZATION OF AMYLASE FROM GERMINATED AFRICAN YAM BEAN SEEDS (Sphenostylis stenocarpa)
The activity of α-amylase and protein concentration in African yam bean seeds increases as germination progresses up to day 8 of germination where it exhibited its highest level, followed by sequenced decreased activity and protein concentration till day 12. Starch from African yam bean seeds, corn and cassava were used for the hydrolysis experiment. There was a significant activity of α-amylase in each of the substrate used, however, the starch from African yam bean seeds had higher α-amylase activity (369.55µmol/min at pH 5.5 and 369.55µmol/min at pH 9.0) followed by starch from corn (367.08µmol/min and 360.49µmol/min at pH 5.5 and 9.0 respectively), while cassava had the least with activity of 353.50µmol/min and 351.03µmol/min at pH 5.5 and 9.0 respectively. The crude enzyme was purified to the level of gel filtration (sephadex G-25) via 80% ammonium sulphate precipitation. The purification fold of 1.36 with specific activity 226.44μmol/min/mg protein and 1.62 with specific activity 367.65μmol/min /mg protein were observed for 80% ammonium sulphate precipitation and gel filtration, respectively. The enzyme displayed optimum activity at pH 5.5 and temperature 45°C in all the three substrates (rAfrican yam bean, corn and cassava). The Michaelis menten constant (Km) and maximum velocity (Vmax) obtained from Lineweaver-Burk plot of initial velocity data at different concentrations of starch from African yam bean seeds as substrate were found to be 0.588mg/ml and 588.24μmol/min, respectively. Similarly, 0.625 mg/ml and 625μmol/min were obtained using starch from corn, respectively, while 0.733mg/ml and 666.7μmol/min were also observed using starch from cassava, respectively. The enzyme activities were enhanced in the presence of some metal ions like Ca2+, Co2+ and Fe2+. Zn2+ and Na2+ neither increased nor decreased enzyme activity while Pb2+ completely inactivated the enzyme.
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