Ceralpha kit

The method of Montanuci et al. [86 (link)] for α- and β-amylases content determination during the malting process was followed. The α- and β-amylase enzymes were determined through the enzymatic Ceralpha kit (K-CERA, Megazyme Southern Cross Rd, Bray, Co. Wicklow, A98 YV29, Ireland) and the enzymatic kit β-amylase (Megazyme, K-BETA3) as detailed in Section 2.4.1 and Section 2.4.2, respectively. All analyses for enzymatic activity were performed in triplicate.

The activity of β-glucosidase was measured by the hydrolysis of FDGlu at 525 nm. Cellulose from T. reesei (Sigma-Aldrich, St. Louis, MO, United States) was used as a standard. For α-amylase activity, ceralpha kit (Megazyme, Bray, Ireland) was used with α-amylase from Aspergillus oryzae (Sigma-Aldrich, St. Louis, MO, United States) as a standard.

One hundred thirty-five μL of polysaccharide solutions were incubated with fifteen μL of protein extracts from germinated grains and germs or purified recombinant GH17 (100 µg/mL). One hundred microliters of aliquot were withdrawn and the amount of reducing ends produced was quantified by Nelson method adapted to microplate [63 ] and read on a microplate reader at a DO of 600 nm. The amount of reducing ends was expressed in μmol/mL or U/mL using standard curve prepared with glucose. Controls were prepared similarly with appropriate buffers and polysaccharide solutions.
α-amylase activities were assayed by using Ceralpha kit (Megazyme) with modifications. Fifty μL of protein extracts from germinated grains were incubated with 50 µL of HR solution (containing pNPG7 substrate and α-glucosidase) for 10 min at 40 °C. The incubation was stopped by adding 0.1 mL of 1 M sodium carbonate solution in an Elisa plate and optical density was measured at 405 nm. Negative control was prepared by replacing protein extract by extracting buffer (citrate phosphate NaCl 1 M pH7).