Binding Assay
Binding assays were performed on the Cel9B mutants using several different substrates: CMC, PASC, BMCC, xylan, xyloglucan and α and β-chitin (Table 3). Assays were performed by incubating a known amount of each mutant protein with each substrate at 4 °C for 1 hr. Then the samples were centrifuged at 6,000 rpm for 10 min and the concentration of unbound protein in the supernatant was determined. By difference to the protein added, the amount of bound protein could be quantitated. Generally, wild type protein and mutants showed stronger affinity to BMCC compared to the other substrates (with the exception of cd9B, and Fn3 and ΔFn3 that showed higher affinity to PASC and xylan, respectively). Wild type Cel9B and mutant M2, were found to have a the highest binding yields of 0.144 and 0.128 on BMCC, respectively. On the other hand, the lowest binding yields were found for mutant ΔE9B, harboring a CBM4, for all the substrates, and cd9B for xylan and α-chitin.
Mutants containing CBM2 showed significantly higher binding yield to PASC than those containing CBM4, a trend that was observed to a smaller extent for xylan. Substrate α-chitin was found to bind to both wild type and M2 (CBM2) at the same yield (approximately 0.104 vs 0.106), followed by M1 (CBM4), in lower amounts. None of the mutants Fn3, E9B or ΔE9B appeared to be associated with any of the substrate fractions. No binding of WT Cel9B or truncation mutants was found for substrates CMC, xyloglucan or β-chitin (data not shown).