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Biochemical and molecular characterization of recombinant acidic and thermostable raw-starch hydrolysing α-amylase from an extreme thermophile Geobacillus thermoleovorans
- Mehta, Deepika, Satyanarayana, T.
- Journal of Molecular Catalysis. B, Enzymatic 2013 v.85-86 pp. 229-238
- Bacillus thermoleovorans, activation energy, alpha-amylase, amino acids, binding sites, calcium, catalytic activity, conserved sequences, corn, enzyme activity, genes, molecular weight, open reading frames, pH, saccharification, signal peptide, starch, temperature, thermal stability, thermophilic microorganisms, wheat
- A gene encoding acidic, thermostable and raw starch hydrolysing α-amylase was cloned from an extreme thermophile Geobacillus thermoleovorans and expressed. The ORF of 1650bp encodes a 515 amino acid protein (Gt-amy) with a signal peptide of 34 amino acids at the N-terminus. Seven conserved sequences of GH-13 family have been found in its sequence. The specific enzyme activity of recombinant Gt-amy is 1723Umg−1 protein with a molecular mass of 59kDa. It is optimally active at pH 5.0 and 80°C with t1/2 values of 283, 184 and 56min at 70, 80 and 90°C, respectively. The activation energy required for its temperature deactivation is 84.96kJmol−1. Ca2+ strongly inhibits Gt-amy at 10mM concentration, and inhibition kinetics with Ca2+ reveals that inhibition occurs as a result of binding to a lower affinity secondary Ca2+ binding site in the active centre in a mixed-type inhibition manner. The Km and kcat of the Gt-amy are 0.315mgmL−1 and 2.62×103s−1, respectively. Gt-amy is Ca2+-independent at the concentration used in industrial starch saccharification, and hydrolyses raw corn and wheat starches efficiently, and thus, is applicable in starch saccharification at the industrial sub-gelatinization temperatures.