Biochemical and molecular characterization of recombinant acidic and thermostable raw-starch hydrolysing α-amylase from an extreme thermophile Geobacillus thermoleovorans
Mehta, Deepika | Satyanarayana, T.
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.Show more [+] Less [-]