Ceralpha α amylase assay kit

Alpha-amylase activity in wheat kernels was evaluated according to the Ceralpha method [37] (link) using Megazyme reagents (Ceralpha α-Amylase Assay Kit) and a detail protocol available at the company website: www.megazyme.com. The same biological and technical sample replicates as for proteome profiling, were applied (Fig. 1). Each technical replicate involved 0.5 g of wheat flour. The enzyme activity was expressed in Ceralpha Units (CU) per gram of flour – one unit of activity was defined as the amount of enzyme, in the presence of excess α-glucosidase and glucoamylase, required to release one micromole of p-nitrophenol from blocked p-nitrophenyl maltoheptaoside (BPNPG7) in one minute under the defined assay conditions. The significance of the differences in amylase activity between the RL and SL was assessed using the Student’s t-test (p≤0.05).

Alpha-amylase activity in triticale kernels was evaluated using the Ceralpha α-Amylase Assay Kit (Megazyme International Ireland Inc., Bray, Ireland) as described in our earlier work on wheat (Perlikowski et al., 2014 (link)).
Beta-amylase activity was tested using the “Betamyl-3® method” Assay Kit (Megazyme International Ireland Ltd., Bray, Ireland). One unit of activity was defined as the amount of enzyme, in the presence of excess thermostable β-glucosidase, required to release one micromole of p-nitrophenol from p-nitrophenyl-β-D-maltotrioside in 1 min under the defined assay conditions.
Three biological and two technical replicates were used (Figure 1). Each technical replicate contain a flour in an amount of 0.5 g. The enzyme activity was shown in Ceralpha Units (CU) per gram of flour and the significance of differences between the RL and SL was assessed using ANOVA (p ≤ 0.05).