GlcNAcase activity assay

GlcNAcase activity was determined by a colorimetric assay using pNP-GlcNAc as substrate. The reaction mixture in a 96-well microtiter plate contained an optimal amount of VhGlcNAcase (0.1 μg for WT and 5 μg for mutants), 500 μM pNP-GlcNAc and 100 mM phosphate buffer, pH 7.0 in a total volume of 200 μL. When sodium formate was added, the final pH in the reaction mixture was found to be 7.0 ± 0.3. The assay was carried out at 37°C with constant agitation in an Eppendorf ThermoMixer^® Comfort (Eppendorf AG, Hamburg, Germany), and was terminated by adding 100 μL of 3 M Na₂CO₃ to each well after 10 min. The concentration of p-nitrophenol (pNP) released was determined at 405 nm in a Biochrom Anthos MultiRead 400 Microplate Reader (Biochrom, Cambridge, UK). The molar quantity of the liberated pNP was calculated from a calibration curve with pNP concentration varied from 0 to 20 nmol. The hydrolytic activity of the enzyme was expressed as the quantity of pNP (nmol) produced in 1 min at 37°C.