Binding Assays
For each enzyme binding assay, 0.2 mg/mL WT Cel9B / each truncation
mutant was mixed separately with excess of substrate (1 mg/mL BMCC,
PASC, CMC, xylan, β-chitin, α-chitin, and xyloglucan) and 0.2 M NaOAc
(pH 5.5) in DNA low bind Eppendorf tubes for a total reaction volume of
160µL . Samples were incubated at 4 °C for 1 hour and run in triplicate.
Enzyme incubated in the absence of substrate or in buffer alone were
included as controls. After incubation, the reaction mixtures were
centrifuged at 4,000 rpm for 5 minutes. Supernatants containing free
protein were mixed into 900 µL Bradford reagent and the concentration
was determined using an Ultraspec 10 (Amersham Biosciences)
spectrophotometer measuring at OD600.