Structure-based engineering of alkaline α-amylase from alkaliphilic Alkalimonas amylolytica for improved thermostability

Abstract

This study aimed to improve the thermostability of alkaline α-amylase from Alkalimonas amylolytica through structure-based rational design and systems engineering of its catalytic domain. Separate engineering strategies were used to increase alkaline α-amylase thermostability: (1) replace histidine residues with leucine to stabilize the least similar region in domain B, (2) change residues (glycine, proline, and glutamine) to stabilize the highly conserved α-helices in domain A, and (3) decrease the free energy of folding predicted by the PoPMuSiC program to stabilize the overall protein structure. A total of 15 single-site mutants were obtained, and four mutants - H209L, Q226V, N302W, and P477V - showed enhanced thermostability. Combinational mutations were subsequently introduced, and the best mutant was triple mutant H209L/Q226V/P477V. Its half-life at 60°C was 3.8-fold of that of the wild type and displayed a 3.2°C increase in melting temperature compared with that of the wild type. Interestingly, other biochemical properties of this mutant also improved: the optimum temperature increased from 50°C to 55°C, the optimum pH shifted from 9.5 to 10.0, the stable pH range expanded from 7.0-11.0 to 6.0-12.0, the specific activity increased by 24%, and the catalytic efficiency (k cat/K m) increased from 1.8×10(4) to 3.5 × 10(4)l/(gmin). Finally, the mechanisms responsible for the increased thermostability were analyzed through comparative analysis of structure models. The structure-based rational design and systems engineering strategies in this study may also improve the thermostability of other industrial enzymes.