Purification and Characterization of Thermostable Α-Amylase by Bacillus Licheniformis RD 24 Isolated from Decayed Refuse at Obafemi Awolowo University Campus, Ile-Ife, Nigeria

Abstract

This study was aimed at the purification and characterization of thermostable α-amylase enzyme produced by thermophilic Bacillus licheniformis RD24 capable of hydrolyzing raw starches. The enzyme was partially purified by 90% ammonium sulphate precipitation and ion exchange chromatography on CM Sepharose CL-6B. The activity was determined, while the protein concentration of the purified α-amylase was estimated by the use of UV Spectrophotometer CECIL CE2502 at 280 nm respectively. The molecular weight of the enzyme was determined using gel filtration on Sephadex G-100. Effects of temperature, pH, metal ions, ethylenediaminetetra acetic acid (EDTA) and kinetic parameters (Km and Vmax) of the purified enzyme were studied. The specific activity of the partially purified Bacillus licheniformis RD24 was determined to be 1.634 Units/mg protein with a purification fold of 4.76. The partially purified Bacillus licheniformis RD24 α-amylase has a molecular weight of 50 kDa. The apparent Vmax and Km obtained with soluble starch for the partially purified Bacillus licheniformis RD24 α-amylase were 4654 ± 108 Units/mg protein and 79.11 ± 1.84 mg/ml. The optimum pH and temperature of partially purified Bacillus licheniformis RD24 α-amylase are 8.0 and 70 o C respectively. Sodium ion has stimulatory effect on the enzyme while Mg 2+ , Ca 2+ and Al 3+ inhibited the enzyme beyond 80 mM, 120 mM and 40 mM respectively. EDTA inhibited the enzyme beyond 20 mM. The study concluded that α-amylase synthesized by Bacillus licheniformis RD24 had a unique characteristic of thermostability and ability to withstand alkaline pH, a much sort after property for various industrial processes.