Characterization of hyperthermostable alpha-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 alpha-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60 degrees C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80 degrees C and pH 5.5-8.5. Maximum enzyme production was in exponential phase with activity 135 U ml(-1) at 60 degrees C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml(-1) at pH 5.0 and 80 degrees 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 micromol mg(-1) protein min(-1), respectively. The amylase was stable over a broad range of temperature from 40 degrees C to 120 degrees C and pH ranges from 5 to 10. The enzyme was stimulated with Mn(2+), whereas it was inhibited by Hg(2+), Cu(2+), Zn(2+), Mg(2+), and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.