The CBM binding assay was performed by mixing 100 μl of CBM (1 mg/ml) in water with 100 μl of CNF (2 mg/ml), followed by incubation at room temperature for 1 hour. To segregate the bound and nonbound CBMs, samples were centrifuged at 21,000g at 22°C for 30 min to pellet the CNF with the bound CBMs and leave the unbound CBMs in the supernatant. The supernatant was discharged, and the pellet was washed twice with 96% ethanol to study whether the CBM binding was stable in the presence of ethanol. Water was used as a control. SDS–polyacrylamide gel electrophoresis was used to analyze the bound and nonbound CBMs.

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