 The study reports functional and structural analyses of TSAGH11 and N01 for xyleinase from thermoanerobacterium sacrolyticum, which is a hemicellulose-degrading bacterium. TSAGH11 was found to be a thermophilic enzyme that favours acidic conditions with maximum activity at pH 5.0 and 70 degrees Celsius. It decomposes xyleins from beechwood and oat speltz to xylose-containing oligosaccharides, with specific activities of 5,622.0 and 3,959.3mg1, respectively. The kinetic parameters, Km and Kcat towards beechwood xylein, are 12.9mgm1 and 34,015.3s1, respectively, resulting in Kcat a Km value of 2,658.7mg1s1, higher by 102 to 103 orders of magnitude compared to other reported GH11s investigated with the same substrate, demonstrating its superior catalytic performance. Crystal structures of TSAGH11 revealed a jelly roll fold, exhibiting open and close conformations of the substrate binding site by distinct conformational flexibility to the thumb region of TSAGH11. In the room temperature structure determined by serial synchrotron crystallography, the electron density map of the thumb domain of the TSAGH11 molecule is disordered, indicating that the thumb domain of TSAGH11 has high structural flexibility at room temperature, with the water molecules in the substrate binding cleft being more disordered than those in the cryogenic structure. These results expand our knowledge of GH11 structural flexibility at room temperature, and pave the way for its application in industrial biomass degradation. This article was authored by Injun Kim, Surin Kim, Kim Hyun Kim, and others. We are article.tv, links in the description below.