Module architecture analysis and application of glycoside hydrolase family 148 thermostable β-1,3-1,4-glucanase

β-1,3-1,4-Glucanases have attracted significant interest because of their potential applications in various food industries. Glycoside hydrolase (GH) family 148 β-1,3-1,4-glucanases exhibit excellent thermal stability and a unique catalytic mechanism, making them particularly promising for certain food processing applications. This study investigated the module architecture enzymatic properties, catalytic mechanisms, and potential applications of a multi-domain β-1,3-1,4-glucanase (EngU) from GH family 148. The overall structure of EngU comprises three independently folding domains, namely the GH148 catalytic domain, CBM4, and the C-terminal domain. The insertion of CBM4 causes the catalytic domain of EngU to be non-contiguous in sequence. The optimum reaction conditions for EngU have been found to be pH 6.0 and 90 °C, demonstrating relatively high thermostability. EngU is an atypical β-1,3-1,4-glucanase, predominantly cleaves the β-1,3 glycosidic bonds in β-1,3-1,4-glucan. Malt saccharification experiments revealed that adding EngU (80 U/g malt) can decreased the viscosity of mash by 6.85 % and the filtration time by 28.83 %. Furthermore, EngU was found to effectively hydrolyze oat bran, producing β-glucooligosaccharides, with the main hydrolysis products being trisaccharide and disaccharide. These results demonstrate the unique module architecture and the application potential of GH family 148 β-1,3-1,4-glucanases, providing valuable insights and resources regarding this category of glycoside hydrolases.