title: Rational design of thermostability in bacterial 1,3-1,4-β-glucanases through spatial compartmentalization of mutational hotspots.
abstract: Higher thermostability is required for 1,3-1,4-β-glucanase to maintain high activity under harsh conditions in the brewing and animal feed industries. In this study, a comprehensive and comparative analysis of thermostability in bacterial β-glucanases was conducted through a method named spatial compartmentalization of mutational hotspots (SCMH), which combined alignment of homologous protein sequences, spatial compartmentalization, and molecular dynamic (MD) simulation. The overall/local flexibility of six homologous β-glucanases was calculated by MD simulation and linearly fitted with enzyme optimal enzymatic temperatures. The calcium region was predicted to be the crucial region for thermostability of bacterial 1,3-1,4-β-glucanases, and optimization of four residue sites in this region by iterative saturation mutagenesis greatly increased the thermostability of a mesophilic β-glucanase (BglT) from Bacillus terquilensis. The E46P/S43E/H205P/S40E mutant showed a 20 °C increase in optimal enzymatic temperature and a 13.8 °C rise in protein melting temperature (Tm) compared to wild-type BglT. Its half-life values at 60 and 70 °C were 3.86-fold and 7.13-fold higher than those of wild-type BglT. The specific activity of E46P/S43E/H205P/S40E mutant was increased by 64.4 %, while its stability under acidic environment was improved. The rational design strategy used in this study might be applied to improve the thermostability of other industrial enzymes.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.|State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214000, China.|School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, China.
authors.forename: Chengtuo
authors.initials: C
authors.lastname: Niu
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.|State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214000, China.|School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, China.
authors.forename: Linjiang
authors.initials: L
authors.lastname: Zhu
authors.affiliation: School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Xu
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China. liqi@jiangnan.edu.cn.|State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214000, China. liqi@jiangnan.edu.cn.|School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, China. liqi@jiangnan.edu.cn.
authors.forename: Qi
authors.initials: Q
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006207:Half-Life; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D006026:Glycoside Hydrolases
keywords: 1,3-1,4-β-Glucanase; Iterative saturation mutagenesis; Molecular dynamics simulation; Spatial compartmentalization; Thermostability