Sucrose Hydrolysis Assay by GmCWI4 Recombinant Protein

GmCWI4 recombinant protein was kindly gifted by Su et al. [40 (link)]. CSVI1 or GmCWI4 protein was incubated with 110–500 mM sucrose (Sangon, Shanghai, China) in 50 mM NaOAc (Sangon, Shanghai, China) buffer, pH 5.0 at 37 °C for different time intervals. After incubation, the reaction was stopped by heating at 95 °C for 5 min. Released fructose and glucose were determined using HPAEC-PAD as described in Wei et al. [60 (link)]. In parallel, glucose and fructose were also determined using a coupled spectrophotometric enzyme assay as described in Link et al. [58 (link)]. All enzyme measurements were performed under conditions in which activities were proportional to enzyme amounts and incubation times.