title: Lysine-Based Site-Directed Mutagenesis Increased Rigid β-Sheet Structure and Thermostability of Mesophilic 1,3-1,4-β-Glucanase.
abstract: 1,3-1,4-β-Glucanase is widely applied in the food industry, while its low thermostability often reduces its performance. In a previous study, chemical modification of surface lysine residues was proved to increase the thermostability of β-glucanase. To improve the thermostability, the mesophilic β-glucanase from Bacillus terquilensis was rationally engineered through site-directed mutagenesis of the 12 lysines into serines. The results showed that the K20S, K117S, and K165S mutants could both enhance the specific activities and thermostability of β-glucanase. The triple mutant (K20S/K117S/K165S) could increase the optimal temperature and T50 value by 15 and 14 °C, respectively. Five percent more structured residues were observed in the mutant, which formed new β-sheet structures in the concave side. Molecular dynamics simulation analysis showed that the flexibility in the mutation regions was decreased, which resulted in the overall rigidity of the β-glucanase. Therefore, the lysine-based site-directed mutagenesis is a simple and effective method for improving the thermostability of β-glucanase.
authors.affiliation: †Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, and ‡Synergetic Innovation Center of Food Safety and Nutrition, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Chengtuo
authors.initials: C
authors.lastname: Niu
authors.affiliation: †Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, and ‡Synergetic Innovation Center of Food Safety and Nutrition, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Linjiang
authors.initials: L
authors.lastname: Zhu
authors.affiliation: †Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, and ‡Synergetic Innovation Center of Food Safety and Nutrition, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Pei
authors.initials: P
authors.lastname: Zhu
authors.affiliation: †Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, and ‡Synergetic Innovation Center of Food Safety and Nutrition, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; D008239:Lysine
keywords: 1,3−1,4-β-glucanase; lysine residues; molecular dynamics simulation; site-directed mutagenesis; thermostability