Real-time monitoring of enzymatic hydrolysis of galactomannans

Enzymatic hydrolysis was monitored in real-time using time dependent static light scattering (TDSLS) for a variety of galactomannans from native Brazi lian flora. alpha -Galactosidase, which strips only the (1-6)alpha -D galac tose side groups, and beta -mannanase, which hydrolyses only the (1-4)beta -D mannan main chain into oligosaccharides were investigated separately and in combination. The time-dependent signatures matched those describing sid e-chain stripping for galactosidase, whereas those resulting from the actio n of mannanase followed the signature typical of random backbone cleavage. Use of both enzymes together required that the TDSLS theory of polymer degr adation be extended to the case where random backbone cleavage sites appear as side chains are stripped by the first enzyme. Whereas galactosidase all owed mannanase to access more backbone cleavage sites as time passes, leadi ng to a higher degree of hydrolysis, there was no increase in rate constant s. The distribution of random fragments in the case of mannanase digestion alone followed reasonably well the predictions for random cleavage of a sin gle-strand polymer with a restricted number of cleavage sites. The fragment distributions were evaluated by size exclusion chromatography. (C) 2001 Jo hn Wiley & Sons. Inc.