4 methylumbelliferyl β d n n diacetylchitobioside hydrate 4mu glcnac 2

Compounds selected by virtual screening were purchased from Topscience (Shanghai, China; http://www.tsbiochem.com) for inhibitory activity assays. The inhibitory activity were assayed in end-point experiments using 4-methylumbelliferyl β-d-N,N'-diacetylchitobioside hydrate (4MU-(GlcNAc)₂, Sigma, St. Louis, MO) as a substrate. The reaction mixture containing 20 mM sodium phosphate buffer (pH 6.0), 1% (v/v) DMSO, 10 nM CeCht1-CAD, 4 μM 4MU-(GlcNAc)₂ and inhibitor was incubated in a final volume of 100 μL at 25 °C for 20 min. The reaction was stopped by adding 100 μL 0.5 M sodium carbonate, and fluorescence of the released 4-MU was quantified (excitation 366 nm, emission 440 nm). Experiments were performed in triplicate unless otherwise specified. The inhibition constant (K_i) was calculated using Dixon plots by changing the compound concentration at several fixed concentrations of 4MU-(GlcNAc)₂ (2 μM, 4 μM, and 8 μM).

Chitinase OfChtI was expressed and purified according to our published literature [18 (link)]. The inhibition assay was carried out following the standard strategy. 4-Methylumbelliferyl β-D-N,N′-diacetylchitobioside hydrate [4MU-(GlcNAc)₂] (Sigma, Shanghai, China) was used as the substrate to calculate the inhibiting activity on OfChtI based on fluorescence intensity, as in a previously reported method [11 (link)].