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

The alpha-amylase (Ba-amy) of Bacillus acidicola was fused with DNA fragments encoding partial N- and C-terminal region of thermostable alpha-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 x 10(4) s(-1) and K (cat)/K (m): 5 x 10(4) mL(-1) mg(-1) s(-1)] and higher thermostability than Ba-amy. The melting temperature (T (m)) of Ba-Gt-amy (73.8 A degrees C) is also higher than Ba-amy (62 A degrees 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 alpha-amylases.