A chimeric α-amylase engineered from Bacillus acidicola and Geobacillus thermoleovorans with improved thermostability and catalytic efficiency

Abstract

Abstract The α-amylase (Ba-amy) of Bacillus acidicola was fused with DNA fragments encoding partial N- and C-terminal region of thermostable α-amylase gene of Geobacillus thermoleovorans (Gt-amy). The chimeric enzyme (Ba-Gt-amy) expressed in Escherichia coli displays marked increase in catalytic efficiency [K  cat: 4 × 104 s−1 and K  cat/K  m: 5 × 104 mL−1 mg−1 s−1] and higher thermostability than Ba-amy. The melting temperature (T  m) of Ba-Gt-amy (73.8 °C) is also higher than Ba-amy (62 °C), and the CD spectrum analysis revealed the stability of the former, despite minor alteration in secondary structure. Langmuir–Hinshelwood kinetic analysis suggests that the adsorption of Ba-Gt-amy onto raw starch is more favourable than Ba-amy. Ba-Gt-amy is thus a suitable biocatalyst for raw starch saccharification at sub-gelatinization temperatures because of its acid stability, thermostability and Ca2+ independence, and better than the other known bacterial acidic α-amylases.