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|Title: ||Biochemical and biophysical characterization of hyperthermophilic sugar isomerases and epimerases|
|Authors: ||Harris, James Morgan|
|Advisors: ||Robert Kelly, Committee Chair|
Jason Haugh, Committee Member
David Ollis, Committee Member
Amy Grunden, Committee Member
|Keywords: ||rare sugars|
|Issue Date: ||3-Aug-2009|
|Discipline: ||Chemical Engineering|
|Abstract: ||The availability of enzymes with optimal functional temperatures above 70Ã‚Â°C has had considerable impact on industrial biocataysis. Extremely thermophilic enzymes have expanded the known thermal range of biological systems and ushered in a new era in applied-biocatalysis less restricted by limitations related to thermoactivity and thermostability. While advances have been in the made in understanding enzyme stabilization at high temperatures, much is to be learned of this complex biomolecular trait. Nonetheless, extremely thermophilic enzymes are being investigated as biocatalysts in a variety of bioprocesses. Here, the biochemical and biophysical properties of hyperthermophilicsugar isomerases and epimerases were examined with respect to their potential to mediate the biosynthesis of monosaccharides with nutritional and medical significance, also know as Ã¢â‚¬Å“rare sugarsÃ¢â‚¬Â .
D-xylose-isomerase from Thermotoga neapolitana 5068 containing an N-terminal fusion with a chitin binding domain (ChiBD) from a hyperthermophilic-chitinase from Pyrococcus furiosus was examined incomparison to the wild type TNXI. The IM ChiBD TNXI half-life (19.9-h) was approximately three times longer than the soluble wild-type TNXI (6.8 h). Furthemore, the unbound soluble ChiBD TNXI had a longer life-life (56.5 h) than the immobilized enzyme. Both unbound and immobilized ChiBD TNXI not only had higher turnover numbers for glucose to fructose than the wild-type enzyme, but also for any known enzyme of this type. Molecular-modeling, based on structural information on the wild-type TNXI and PfChiBD, showed that the N-terminal fusion likely impacted subunit interactions, thereby contributing to the enhanced thermostability of the unbound ChiBD TNXI. These results illustrate that substantial changes in thermostability and reaction-kinetics can result from affinity tags for hyperthermophilic proteins.
Sugar isomerases and epimerases (L-fucose isomerase (TMFI), L-arabinose isomerase TMAI), L-rhamnose isomerase (TMRI), D-tagatose-3-epimerase (TMTE), and D-xylose isomerase (TNXI)) from the hyperthermophilic bacterial genus Thermotogaawere examined as biocatalysts for rare sugar synthesis. Single and multi step reactions involving each isomerase with TMTE produced both expected and unexpected products, based on similar experiments with homologous mesophilic enzymes. The recently reported TMTE three-dimensional-structure revealed a non-conserved active site and hydrophobic binding pocket compared to mesophilic epimerases, likely responsible for the biocatalyic results observed in this study.|
|Appears in Collections:||Dissertations|
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