The primary purpose of this study was to (1) develop methods for the
analysis of and (2) provide information on the chemical nature of reducing ends in
typical cellulose substrates used for the study of cellulolytic enzymes. The studies
were designed such that values obtained for cellulose substrates were compared
with those obtained for a series of soluble cellooligosaccharides. The initial phase
of the study tested the validity of using established colorimetric reducing sugar
assays, developed for the measurement of reducing sugars in solution, for the
quantification of reducing ends on insoluble substrates. The results demonstrate
that published methods give widely differing values for the number of reducing
ends per unit weight cellulose. The Cu⁺⁺-based assay, using bicinchoninic acid
(BCA) as a color yielding chelator of Cu⁺, is shown to provide values that appear
most consistent the properties of the substrates. A method was developed using
the Cu⁺⁺-BCA reagent, following a mild sodium borohydride treatment, to provide
an estimate of the number of solvent accessible reducing ends on insoluble
substrates. The kinetics of sodium borohydride reduction of reducing ends on
crystalline cellulose, amorphous cellulose and soluble cellooligosaccharides were
compared in order to ascertain the relative reactivity of these reducing ends. The
apparent second order rate constants for the reduction of reducing ends associated
with the crystalline celluloses were significantly lower than those for the reduction
of reducing ends associated with either the insoluble amorphous celluloses or the
soluble cellooligosaccharides. These results indicate the reducing ends associated
with crystalline celluloses are not extended out from the surface as though
mimicking solution phase reducing ends. The relevance of this, as well as the
other results, to the behavior of cellulolytic enzymes is discussed. The final phase
of the study was the demonstration of both a reducing sugar-based and a viscositybased
assay for the detection of a prototypical polysaccharide depolymerizing
glycosyl hydrolase, polygalacturonase. / Graduation date: 2004
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/29899 |
| Date | 28 October 2003 |
| Creators | Kongruang, Sasithorn |
| Contributors | Penner, Michael H., Bolte, John P. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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