title: Structure-based rational design and introduction of arginines on the surface of an alkaline α-amylase from Alkalimonas amylolytica for improved thermostability.
abstract: In this study, the thermostability of an alkaline α-amylase from Alkalimonas amylolytica was significantly improved through structure-based rational and the introduction of multiple arginines (Arg) on the protein surface. Based on an analysis of the tertiary structure, seven residues (glutamine (Gln) 166, Gln 169, serine (Ser) 270, lysine (Lys) 315, Gln 327, asparagine (Asn) 346, and Asn 423) were selected as engineering targets and individually replaced with arginine. Five of the seven single-mutated enzymes-S270R, K315R, Q327R, N346R, and N423R-showed enhanced thermostability. Multiple arginines were subsequently introduced on the protein surface, and the quintuple-mutated enzyme S270R/K315R/Q327R/N346R/N423R showed a 6.4-fold improvement in half-life at 60 and a 5.4 °C increase in melting temperature (T m) compared with those of wild-type enzyme. Concomitantly, the optimal temperature, optimal pH, and catalytic efficiency of this mutated enzyme also improved. The mutated enzyme displayed a large shift in optimal pH from 9.5 to 11.0. In addition, the optimum temperature increased from 50 to 55 °C, and the catalytic efficiency (k cat/K m) increased from 1.8 × 10(4) to 3.6 × 10(4) L/(g · min). The intramolecular interactions of mutated enzymes that contributed to increased thermostability were examined through comparative analysis of the model structures of wild-type and mutated enzymes. The thermostable mutated enzymes generated in this study have potential applications in the textile industry.
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: Hyun-dong
authors.initials: HD
authors.lastname: Shin
authors.forename: Jianghua
authors.initials: J
authors.lastname: Li
authors.forename: Long
authors.initials: L
authors.lastname: Liu
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics; D004795:Enzyme Stability; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D044366:Transition Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D001120:Arginine; D000516:alpha-Amylases