title: 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/(g min). 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.
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.
authors.forename: Zhuangmei
authors.initials: Z
authors.lastname: Deng
authors.forename: Haiquan
authors.initials: H
authors.lastname: Yang
authors.forename: Jianghua
authors.initials: J
authors.lastname: Li
authors.forename: Hyun-Dong
authors.initials: HD
authors.lastname: Shin
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D019943:Amino Acid Substitution; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D000516:alpha-Amylases