Biolayer interferometry binding studies
The ForteBio Octet® RED96 interaction analyzer(39-41) (ForteBio, United States) was used to detect the binding of XynA to the oligosaccharide substrates (cellobiose, xylohexaose and cellohexaose) (Megazyme, Ireland) and xylan (BWX) (Shanghai, China) (Li, Tian, Wang, Zhu, & Sun, 2017; Petersen, 2017). First, XynA was dialyzed against PBS. Second, the equivalent biotinylation kit (Biotinylation Kit, Jiangsu Bomeida Life Science Co., Ltd.) was added to bind XynA protein for 60 minutes for biotinylating. Finally, the unbound biotin was removed with a desalination column. Before the experiment, we wetted the biosensor (SSA, Shenzhen Baokeda Biotechnology Co., Ltd.) with PBS for 10 minutes. In the first step, we equilibrated the sensor with PBS for 2 minutes. In the second step, XynA was loaded onto the SSA sensor until it was saturated. Third, we equilibrated with PBS for 3 minutes. Fourth, diluted substrate solutions (12.5, 25, 50, 100, 200, and 250 μM) were sequentially added and dissociated from low concentration to high concentration, and each combination and dissociation time was 300 seconds. A sensor not bound to the biotinylated XynA was used as a baseline control for calibration. The whole process was carried out at room temperature, and all tests were repeated three times.