Purification, immobilization and characterization of thermostable α-amylase from a thermophilic bacterium Geobacillus sp TF14

Abstract

Objective: In this study, alpha-amylase from a thermophilic bacterium Geobacillus sp. TF14 was purified and immobilized on two different supports. Methods: Ion exchange and hydrophobic interaction chromatography techniques were employed for the purification. Results: The enzyme was purified as 17.11 fold and determined as a single band of 54 kDa on SDS-PAGE. Purified enzyme showed two pH optimums of pH 5.00 and pH 9.00 and the enzyme is quite stable at these pHs over a period of 48 h. Purified enzyme showed maximal activity at 75 degrees C and stability at this temperature over a period of 72 h. It was observed that Ca2+ activated the enzyme at about 70% at 5 mM final concentration. SDS, Triton X100, Triton X114 and Tween 20 caused around 50% loss of initial activity at a final concentration of 1% (w/v). Purified enzyme was immobilized on the surface of Dowex and chitin. Immobilization highly enhanced temperature optima and thermal stability. Dowex immobilized enzyme maintained most of its initial activity in the presence of SDS, Triton X100, Triton X114 and Tween 20 at a concentration of 1%. Conclusion: It can be concluded that the purified enzyme may find application in many fields of starch based industries.