We have attempted to understand the mechanisms of indigo backstaining during enzyme stone washing by separately analyzing dye staining levels on different cotton surfaces during the process. The high ability of the cellulase enzyme protein to bind to cotton cellulose is the major cause of backstaining. Further studies of commercial cellulases show that the pH of maximum cellulase activity may not be the same pH of maximum of cellulase binding capacity to the substrate, and also that indigo dyes have completely different affinities for cellulase proteins from different fungal origins.
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