ISOLATION, PARTIAL PURIFICATION AND CHARACTERIZATION OF α-AMYLASE FROM Bacillus alcalophilus
The aim of this study was to isolate, partially purify and characterize α-Amylase from Bacillus alcalophilus. The enzyme (α-amylase) was isolated from Bacillus alcalophilus using cassava as only carbon source. 20 g of soil sample were weighed out and dissolved in 40 ml of distilled water in a clean conical flask, mixed vigorously and heated at 60oC for 60 min in water bath and taken as the stock culture. From the stock preparation, ten folds serial dilution were carried out and the 10-4 to 10-6 dilutions were plated out in a media plate. Percentage ammonium sulphate saturation, ammonium sulphate precipitation and gel filtrations were carried out to partially purify α-amylase from Bacillus alcalophilus. The α-amylase was then characterised by studying the effect of pH, change in temperature, substrate concentration and metal ion on the enzyme`s activity. The specific activity of the crude enzyme was 160.26 U/mg proteins. After ammonium sulphate precipitation and gel filtration, the specific activity were found to be 88.9 and 285.9U/mg protein, respectively. The optimum pH was found to be 7.5 and 70°C respectively. The α-amylase activity was found to be enhanced by Ca2+, Mg2+, Mn2+and mCo2+, whereas Fe2+ was found to have inhibitory effect. The enzyme retained more than 80% of its activity at 60 min in the presence of Ca2+, Mg2+ , Mn2+and Co2+, and lost up to 90% of its activity in the presence of Fe2+. In this study, Ca2+ maintained more stability of the enzyme than all other metal ions. The Michaelis-Mentens constant (Km) and maximum velocity (Vmax) obtained from the Line Weaver Burk plot of initial velocity data of different substrate concentrations were 1.159 mg/mL and 16.24 μmol/min respectively. In conclusion, this study revealed the potentials Bacillus alcalophilus to serve as other source of α-amylase, especially for industrial purposes.
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