Production, purification and characterization of thermostable alpha amylase from Bacillus subtilis Y25 isolated from decaying yam (Dioscorea rotundata) tuber

Abstract

Amylases have wide biotechnological potentials for applications in various industries. An α-amylase-producing bacterium was isolated from deteriorating yam tubers. Molecular characterization using the 16S rRNA gene sequencing was used to confirm the identity of the bacterium as Bacillus subtilis Y25. The effect of some cultural and nutritional factors such as pH, temperature, carbon and nitrogen sources on α-amylase production from the bacterium was determined. Maximum α-amylase production was observed using starch and peptone as carbon and nitrogen sources, respectively, with an initial medium pH of 8.0 and incubation at 45 °C for 36 h. The enzyme was purified by ion exchange chromatography on CM Sepharose CL-6B. The kinetic parameters Km and Vmax of the enzyme, as well as the effect of pH, temperature, metal ions and ethylenediaminetetra acetic acid (EDTA) on the activity of the purified enzyme were studied. The specific activity of the partially purified enzyme was determined to be 15.21 Units/mg protein with a purification fold of 3.80. The molecular weight of the purified enzyme was estimated to be 58.0 kDa. The Vmax and Km values obtained with soluble starch for Bacillus subtilis Y25 α-amylase were 314.10 ± 23.30 Units/mg protein and 53.98 ± 12.03 mg/ml, respectively. The enzyme exhibited optimum activity at a temperature of 60 °C and pH 8.0. The metal ion Ca2+ had no effect on the enzyme at 20 mM concentration, whereas Na+ and Mg2+, as well as EDTA inhibited the enzyme at the same concentration. The characteristics of the α-amylase from Bacillus subtilis Y25 revealed it to be a thermostable and an alkaline metalloenzyme with potential for applications in the detergent and saccharification industries.