Characterization of hyperthermostable α-amylase from Geobacillus sp. IIPTN

A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum enzyme production was in exponential phase with activity 135 U ml⁻¹ at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml⁻¹ at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg⁻¹ protein. The molecular mass of the purified enzyme was 97 KDa. The values of K _m and V _max were 36 mg ml⁻¹ and 222 μmol mg⁻¹ protein min⁻¹, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The enzyme was stimulated with Mn²⁺, whereas it was inhibited by Hg²⁺, Cu²⁺, Zn²⁺, Mg²⁺, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.