Degree Type


Date of Award


Degree Name

Doctor of Philosophy


Chemical and Biological Engineering

First Advisor

Peter J. Reilly


The subsite affinity map of an endo-(1 → 4)-[beta]-xylanase produced by Aspergillus niger has been determined by using linear, [superscript]3H-reducing-end-labeled xylooligosaccharides ranging in length from xylotriose through xylooctaose. No evidence of multiple attack or of condensation and transxylosylation reactions was found. Bond cleavage frequencies were highest near the reducing end of short substrates, with the locus of highest frequencies moving towards the middle of larger substrates. This endoxylanase has five major subsites, with the catalytic site located between the third and fourth subsites, counting from the non-reducing end of the bound substrate. The subsite to the non-reducing side of the catalytic site strongly repels its corresponding D-xylosyl residue, characteristic of endohydrolases that produce mainly oligosaccharides but do not catalyze bi-substrate reactions. The results demonstrate that this endoxylanase is similar in the structure of its active center to others previously subsite-mapped;These results were compared to similar studies on dextransucrase with the same inhibitors. The K[subscript] M value of sucrose is 26.5 ± 4.6mM. Derivatives modified at the C6-position were potent competitive inhibitors, with K[subscript] i values of 6.2 ± 0.3mM (6-deoxysucrose) and 0.50 ± 0.06mM (6-deoxy-6-fluorosucrose). Sucroses modified at the 3-position were not significantly inhibitory over the concentration range tested. 4,6-Dideoxysucrose gave an unusual non-competitive inhibition, in that increasing its concentration did not produce a commensurate increase in the level of inhibition, which instead appeared to approach a limit. None of these sucrose derivatives were substrates for amylosucrase, nor were they glycosyl donors to maltotriose;These results show remarkable similarity to results obtained for dextransucrase, implying that the binding structures may be highly similar. Since the basic catalytic function of both enzymes is the formation of [alpha]-glycosidic bonds, albeit to different hydroxyl moieties on the glucose ring, it would appear that the substrate binding structure may be the determining factor in the product polysaccharide structure;The catalytic mechanism of porcine pancreatic [alpha]-amylase was examined by nuclear magnetic resonance (n.m.r.) at subzero temperatures using (1-[superscript]13C) -labeled maltotetraose. Using spectral summation and difference techniques, a broad resonance peak was detected corresponding to a [beta]-carboxyl-acetal ester covalent enzyme-substrate intermediate. This evidence supports the covalent mechanism hypothesis for porcine pancreatic [alpha]-amylase, based on the presumption of the catalytic capacity of aspartic acid residues in the active site of this enzyme.



Digital Repository @ Iowa State University,

Copyright Owner

Bernard Yi Tao



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109 pages