Examining the molecular characteristics of glycoside hydrolase family 20 β-N-acetylglucosaminidases with high activity

β-N-Acetylglucosaminidases (GlcNAcases) possess many important biological functions and are used for promising applications that are often hampered by low-activity enzymes. We previously demonstrated that most GlcNAcases of the glycoside hydrolase (GH) family 20 showed higher activities than those of other GH families, and we presented two novel GH 20 GlcNAcases that showed higher activities than most GlcNAcases. A highly flexible structure, which was attributed to the presence of to a high proportion of random coils and flexible amino acid residues, was presumed to be a factor in the high activity of GH 20 GlcNAcases. In this study, we further hypothesized that two special positions might play a key role in catalytic activity. The increase in GH 20 GlcNAcase activity might correspond to the increased structural flexibility and substrate affinity of the two positions due to an increase in random coils and amino acid residues, notably acidic Asp and Glu.