Biochemical and molecular characterization of recombinant acidic and thermostable raw-starch hydrolysing α-amylase from an extreme thermophile Geobacillus thermoleovorans

A gene encoding acidic, thermostable and raw starch hydrolysing a-amylase was cloned from an extreme thermophile Geobacillus thermoleovorans and expressed. The ORF of 1650 bp 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 1723 U mg(-1) protein with a molecular mass of 59 kDa. It is optimally active at pH 5.0 and 80 degrees C with t(1/2) values of 283, 184 and 56 min at 70,80 and 90 degrees C, respectively. The activation energy required for its temperature deactivation is 84.96 kJ mol(-1). Ca2+ strongly inhibits Gt-amy at 10 mM 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 K-m and k(cat) of the Gt-amy are 0.315 mg mL(-1) and 2.62 x 10(3) s(-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. (C) 2012 Elsevier B.V. All rights reserved.