KINETICS AND THERMODYNAMIC STUDIES OF ALPHA AMYLASE FROM BACILLUS LICHENIFORMIS MUTANT

Abstract

The present investigation deals with the purification and characterization of enzyme α-amylase from a mutant strain of Bacillus licheniformis EMS-6. A laboratory scale stirred fermentor of 7.5 L capacity was used for the enzyme production under optimal conditions. The enzyme was purified up to homogeneity level by Ammonium sulphate and ion-exchange chromatography using a fast protein liquid chromatography (FPLC) system. The specific activity of the enzyme increased 4-5 times while the yield was found to be 40.4%. The purification fold by RESOURCE-S was recorded to be 3.58. The molecular weight was found to be 55 KDa. In the present research work, the Vmax (2778 U/mg/min) and Km (8.3mg/ml) of α-amylase were derived from the Lineweaver Burke plot. Thermodynamic parameters for soluble starch hydrolysis, Ea, ΔH, ΔS and ΔG of α-amylase from B. licheniformis EMS-6 were found to be 25.14 KJ/mol, 22.53 KJ/mole, -110.95J/mole/K and 36968 J/mole, respectively. The enzyme was stable over a pH range of 4.5-9.0 and gave pH optimum of 7.0. The pKa1 and pKa2 of ionizable groups of active site controlling Vmax, determined by Dixon plot, were 6.0 and 7.5, respectively.