Activity Assays
Activity assays were performed in triplicate as follows: 2-fold
decreasing concentrations of WT Cel9B enzyme and truncation mutants were
incubated for various times (16 hours for BMCC, PASC and xylan, 2 hours
for CMC, and 30 minutes for xyloglucan) with 1 mg/ mL of the substrates
at 50°C. After each incubation period, the amount of glucose released
was determined by PAHBAH assay as described in Kruer-Zerhusen et al.,
2017. Briefly, 40 µL of each sample was mixed in a 96 well plate with
120 µL PAHBAH solution (0.5% 4-hydroxy-benzhydrazide in 0.5 M HCl and
0.5 M NaOH) as The plate was then sealed and heated to 100°C for 6
minutes. Plates were cooled to 42°C and the glucose concentration was
measured using a microplate spectrophotometer at 410 nm (Synergy 2,
BioTek Instruments Inc., Winooski, VT and a standard curve previously
generated.