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

• Published in: Journal of molecular catalysis. B, Enzymatic Enzymatic, 2013-01, Vol.85-86, p.229-238
• Main Authors: Mehta, Deepika, Satyanarayana, T.
• Format: Article
• Language: English
• Subjects: activation energy; alpha-amylase; amino acids; Bacillus thermoleovorans; binding sites; Bioconversions. Hemisynthesis; Biological and medical sciences; Biotechnology; Ca2+-independent/inhibited α-amylase; calcium; catalytic activity; conserved sequences; corn; enzyme activity; Fundamental and applied biological sciences. Psychology; genes; Geobacillus thermoleovorans; Methods. Procedures. Technologies; molecular weight; open reading frames; Raw-starch hydrolysis; Recombinant Gt-amy; saccharification; signal peptide; starch; temperature; thermal stability; thermophilic microorganisms; Thermostable α-amylase; wheat
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2012.08.017

[Display omitted] ► Expressed a gene encoding acidic thermostable α-amylase from G. thermoleovorans. ► This is the first ever reported Ca2+-inhibited acidic α-amylase. ► Biochemical characterization of the recombinant enzyme (Gt-amy). ► Gt-amy is Ca2+-independent at the concentration used in starch saccharification. ► Gt-amy saccharifies raw starches. 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.