Determination of the native features of the exoglucanase Cel48S from Clostridium thermocellum

摘要: Clostridium thermocellum is considered one of the most efficient natural cellulose degraders because its cellulosomal system. As major exoglucanase cellulosome in C. thermocellum, Cel48S plays key roles and influences activity features to a great extent. Thus, it importance reveal enzymatic Cel48S. However, has not been well performed due difficulties purifying either recombinant or native proteins. We observed that soluble fraction catalytic domain (Cel48S_CD) obtained by heterologous expression Escherichia coli denaturation-refolding treatment contained large portion incorrectly folded proteins with low activity. Using previously developed seamless genome-editing system for we achieved direct purification Cel48S_CD from culture supernatant DSM1313 inserting sequence encoding 12 successive histidine residues TAA stop codon immediately behind GH Based on fully active protein, biochemical structural analyses were innate characteristics. The showed high 117.61 ± 2.98 U/mg apparent substrate preference crystalline under assay conditions. crystal structure GH48 protein revealed substrate-coupled changes residue conformation, indicating induced-fit effects between substrates. Mass spectrum suggested no significant posttranslational modification protein. Our results confirmed specificity consistent cellulosome. evidence conformational during binding. In addition, our study provided reliable method situ other secretive thermocellum.