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

• Published in: Journal of industrial microbiology & biotechnology 2016-04, Vol.43 (4), p.473-484
• Main Authors: Parashar, Deepak, Satyanarayana, T
• Format: Article
• Language: English
• Subjects: adsorption; alpha-amylase; alpha-Amylases - chemistry; alpha-Amylases - genetics; alpha-Amylases - metabolism; Bacillus; Bacillus - enzymology; Bacillus - genetics; Bacillus thermoleovorans; Biocatalysis; biocatalysts; Biochemistry; Bioinformatics; Biomedical and Life Sciences; Biotechnology; Biotechnology Methods; calcium; catalytic activity; chimerism; DNA fragmentation; Enzyme Stability - genetics; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; genes; Genetic Engineering; Geobacillus; Geobacillus - enzymology; Geobacillus - genetics; Hydrogen-Ion Concentration; Inorganic Chemistry; Kinetics; Life Sciences; melting point; Microbiology; Mutant Chimeric Proteins - chemistry; Mutant Chimeric Proteins - genetics; Mutant Chimeric Proteins - metabolism; Protein Structure, Secondary - genetics; saccharification; starch; Starch - metabolism; Temperature; thermal stability
• ISSN: 1367-5435; 1476-5535
• DOI: 10.1007/s10295-015-1721-7

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.