Journal
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
Volume 1865, Issue 10, Pages 1237-1245Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbapap.2017.06.015
Keywords
Amylase; Thermotoga petrophila; Crystal structure; Dimerization; Cloning; Expression; Purification
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Funding
- Foreign Fellowship (International Research Support Initiative Program) [I-/HEC/HRD/2012/2310. PIN: IRSIP 21BMS 27]
- Higher Education Commission of Pakistan
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Thermostable alpha-amylases have many industrial applications and are therefore continuously explored from novel sources. We present the characterization of a novel putative alpha-amylase gene product (Tp-AmyS) cloned from Thermotoga petrophila. The purified recombinant enzyme is highly thermostable and able to hydrolyze starch into dextrin between 90 and 100 degrees C, with optimum activity at 98 degrees C and pH 8.5. The activity increased in the presence of Rbl +, K1+ and Ca2+ ions, whereas other ions inhibited activity. The crystal structure of Tp-AmyS at 1.7 angstrom resolution showed common features of the GH-13 family, however was apparently found to be a dimer. Several residues from one monomer interacted with a docked acarbose, an inhibitor of Tp-AmyS, in the other monomer, suggesting catalytic cooperativity within the dimer. The most striking feature of the dimer was that it resembled the dimerization of salivary amylase from a previous crystal structure, and thus could be a functional feature of some amylases.
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