Biophysical and biochemical characterization of a hyperthermostable and Ca2+ -independent alpha-Amylase of an extreme thermophile Geobacillus thermoleovorans.
alpha-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 alpha-amylase (26 kDa; alpha-amylase gt), which is hyperthermostable (Topt 100 degrees C) and does not require Ca2+ for its activity/stability. These special features of alpha-amylase gt make it applicable in starch saccharification process. The structural aspects of alpha-amylase gt are, therefore, of significant interest to understand its structure-function relationship. The circular dichroism spectroscopic data revealed the native alpha-amylase gt to contain 25% alpha-helix, 21% beta-sheet, and 54% random coils. The addition of urea, at high concentration (8 M), appeared to expose the buried Trp residues of the native alpha-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 Ksv and fa values of KI, CsCl, and acrylamide suggested the overall Trp microenvironment in alpha-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 alpha-helix content or the enzyme activity with an increase in temperature (60-100 degrees C) that suggested a critical role of the alpha-helix content in maintaining the catalytic activity.
Uma Maheswar Rao
J L
JL
Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India.
Satyanarayana
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D001406:Bacillaceae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D004795:Enzyme Stability; D013696:Temperature; D000516:alpha-Amylases / Q000378:metabolism*
D000516:alpha-Amylases; D002118:Calcium