Glycoside Hydrolase Substrate Specificity

In order to analyze the hydrolysis substrate specificity of the DogH glycoside hydrolase, nine carbohydrates with different glycosidic bonds were selected for analysis in this study, as listed in Table 2. According to [17 (link)], the method of Benedict’s experiment was used to detect the reducing sugar content of the hydrolyzed substrates. Benedict’s experiment was conducted for a range of concentrations of glucose and maltose to confirm the differences between mono- and disaccharides and to determine reasonable concentrations for the main analysis; the standard curves are shown in Supplementary Figure S6. The final concentrations of reducing and non-reducing sugars were 0.25% and 0.5%, respectively. Amylase decomposition of starch was used as a positive control. Moreover, 200 μg/mL of DogH and an equivalent buffer solution without DogH were added to the solution and incubated at 37 °C for 4 h. The reaction was subsequently terminated by the addition of 500 μL of Benedict’s experiment and incubated at 95 °C for 10 min. The absorbance value of each sample was measured at 320 nm to determine the extent of copper reduction in the substrate. Three replicates of each carbohydrate were measured in the presence and absence of DogH addition.