Biophysical and Biochemical Characterization of a Hyperthermostable and Ca²⁺-independent α-Amylase of an Extreme Thermophile Geobacillus thermoleovorans

α-Amylases reported from various microbial sources have been shown to be moderately thermostable and Ca²⁺ dependent. The bacterial strain used in this investigation is an extremely thermophilic bacterium Geobacillus thermoleovorans that produces a novel α-amylase (26 kDa; α-amylase gt), which is hyperthermostable (T(opt) 100 °C) and does not require Ca²⁺ for its activity/stability. These special features of α-amylase gt make it applicable in starch saccharification process. The structural aspects of α-amylase gt are, therefore, of significant interest to understand its structure-function relationship. The circular dichroism spectroscopic data revealed the native α-amylase gt to contain 25% α-helix, 21% β-sheet, and 54% random coils. The addition of urea, at high concentration (8 M), appeared to expose the buried Trp residues of the native α-amylase gt to the aqueous environment and thus showed low fluorophore. Fluorescence-quenching experiments using KI, CsCl, N-bromosuccinimide, and acrylamide revealed interesting features of the tryptophan microenvironment. Analysis of K(sv) and f(a) values of KI, CsCl, and acrylamide suggested the overall Trp microenvironment in α-amylase to be slightly electropositive. Fluorescence-quenching studies with acrylamide revealed the occurrence of both collisional as well as static quenching processes. There was no change in the α-helix content or the enzyme activity with an increase in temperature (60-100 °C) that suggested a critical role of the α-helix content in maintaining the catalytic activity.