The role of N185D substitution in enhancing activity of BaqA∆C α-amylase

Abstract

Abstract α-Amylases are endoenzymes capable of degrading starch into branched and linear oligosaccharides. Since starch-processing industries rely on starch-rich mashes, exploring amylases that can efficiently hydrolyse highly concentrated raw starch is crucial. BaqA from Bacillus aquimaris MKSC 6.2 is particularly promising as it hydrolyses raw starch into simple sugars without preheating, offering potential energy and cost savings for industrial applications. This study investigates the functional role of a specific residue within Conserved Sequence Region (CSR) V of BaqAΔC (a truncated BaqA lacking 34 amino acids from the C terminus), predicted to influence catalytic activity, product specificity, and substrate binding. A BaqA∆CN185D mutant was generated through site-directed mutagenesis and expressed in Escherichia coli ArcticExpress (DE3). Compared with BaqAΔC, the mutant displayed higher specific activity (7.6 ± 0.2 vs. 5.8 ± 0.4 U/mg) and a greater degree of hydrolysis (2.18% vs. 1.41%). Futhermore, BaqAΔCN185D showed a significantly lower K m (9.48 ± 1.3 mg/mL) compared to BaqAΔC (23.48 ± 4.8 mg/mL), reflecting higher substrate affinity. Its catalytic efficiency ( k ca. t / K m ) also increased from 5.65 ± 0.5 to 9.8 ± 0.9 mL·mg⁻¹·s⁻¹, indicating enhanced substrate binding and overall catalytic performance due to the N185D mutation. HPLC analysis of crude protein extracts from E. coli ArcticExpress (DE3) expressing BaqA∆C and BaqA∆CN185D revealed saccharifying activity, whereas the control strain produced a native liquefying α-amylase. Molecular dynamics simulations further indicated that the N185D substitution altered catalytic and substrate-binding site residues, likely creating a more effective substrate-binding gateway. Collectively, these findings highlight the critical role of N185 in modulating BaqA∆C’s catalytic efficiency and product profile, providing valuable insights for industrial enzyme engineering.