title: Distinct roles for carbohydrate-binding modules of glycoside hydrolase 10 (GH10) and GH11 xylanases from Caldicellulosiruptor sp. strain F32 in thermostability and catalytic efficiency.
abstract: Xylanases are crucial for lignocellulosic biomass deconstruction and generally contain noncatalytic carbohydrate-binding modules (CBMs) accessing recalcitrant polymers. Understanding how multimodular enzymes assemble can benefit protein engineering by aiming at accommodating various environmental conditions. Two multimodular xylanases, XynA and XynB, which belong to glycoside hydrolase families 11 (GH11) and GH10, respectively, have been identified from Caldicellulosiruptor sp. strain F32. In this study, both xylanases and their truncated mutants were overexpressed in Escherichia coli, purified, and characterized. GH11 XynATM1 lacking CBM exhibited a considerable improvement in specific activity (215.8 U nmol(-1) versus 94.7 U nmol(-1)) and thermal stability (half-life of 48 h versus 5.5 h at 75°C) compared with those of XynA. However, GH10 XynB showed higher enzyme activity and thermostability than its truncated mutant without CBM. Site-directed mutagenesis of N-terminal amino acids resulted in a mutant, XynATM1-M, with 50% residual activity improvement at 75°C for 48 h, revealing that the disordered region influenced protein thermostability negatively. The thermal stability of both xylanases and their truncated mutants were consistent with their melting temperature (Tm), which was determined by using differential scanning calorimetry. Through homology modeling and cross-linking analysis, we demonstrated that for XynB, the resistance against thermoinactivation generally was enhanced through improving both domain properties and interdomain interactions, whereas for XynA, no interdomain interactions were observed. Optimized intramolecular interactions can accelerate thermostability, which provided microbes a powerful evolutionary strategy to assemble catalysts that are adapted to various ecological conditions.
authors.affiliation: Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Dong-Dong
authors.initials: DD
authors.lastname: Meng
authors.affiliation: Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Ying
authors.affiliation: Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China.
authors.forename: Xiao-Hua
authors.initials: XH
authors.lastname: Chen
authors.affiliation: Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Lu
authors.affiliation: Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China.
authors.forename: Kang
authors.initials: K
authors.lastname: Ning
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, China.
authors.forename: Lu-Shan
authors.initials: LS
authors.lastname: Wang
authors.affiliation: Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China lifl@qibebt.ac.cn.
authors.forename: Fu-Li
authors.initials: FL
authors.lastname: Li
mesh_terms: D002151:Calorimetry; D050260:Carbohydrate Metabolism; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D000068536:Bacillota / Q000201:enzymology*; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D044366:Transition Temperature
chemical_list: D011994:Recombinant Proteins; D006026:Glycoside Hydrolases