@article{harris_epting_kelly_2010, title={N-terminal Fusion of a Hyperthermophilic Chitin-Binding Domain to Xylose Isomerase from Thermotoga neapolitana Enhances Kinetics and Thermostability of Both Free and Immobilized Enzymes}, volume={26}, ISSN={["1520-6033"]}, DOI={10.1002/btpr.416}, abstractNote={AbstractImmobilization of a thermostable D‐xylose isomerase (EC 5.3.1.5) from Thermotoga neapolitana 5068 (TNXI) on chitin beads was accomplished via a N‐terminal fusion with a chitin‐binding domain (CBD) from a hyperthermophilic chitinase produced by Pyrococcus furiosus (PF1233) to create a fusion protein (CBD‐TNXI). The turnover numbers for glucose to fructose conversion for both unbound and immobilized CBD‐TNXI were greater than the wild‐type enzyme: kcat (min−1) was ∼1,000, 3,800, and 5,800 at 80°C compared to 1,140, 10,350, and 7,000 at 90°C, for the wild‐type, unbound, and immobilized enzymes, respectively. These kcat values for the glucose to fructose isomerization measured are the highest reported to date for any XI at any temperature. Enzyme kinetic inactivation at 100°C, as determined from a bi‐phasic inactivation model, showed that the CBD‐TNXI bound to chitin had a half‐life approximately three times longer than the soluble wild‐type TNXI (19.9 hours vs. 6.8 hours, respectively). Surprisingly, the unbound soluble CBD‐TNXI had a significantly longer half‐life (56.5 hours) than the immobilized enzyme. Molecular modeling results suggest that the N‐terminal fusion impacted subunit interactions, thereby contributing to the enhanced thermostability of both the unbound and immobilized CBD‐TNXI. These interactions likely also played a role in modifying active site structure, thereby diminishing substrate‐binding affinities and generating higher turnover rates in the unbound fusion protein. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010}, number={4}, journal={BIOTECHNOLOGY PROGRESS}, author={Harris, James M. and Epting, Kevin L. and Kelly, Robert M.}, year={2010}, pages={993–1000} }
 @article{comfort_bobrov_ivanen_shabalin_harris_kulminskaya_brumer_kelly_2007, title={Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases}, volume={46}, ISSN={["0006-2960"]}, DOI={10.1021/bi061521n}, abstractNote={Organization of glycoside hydrolase (GH) families into clans expands the utility of information on catalytic mechanisms of member enzymes. This issue was examined for GH27 and GH36 through biochemical analysis of GH36 alpha-galactosidase from Thermotoga maritima (TmGalA). Catalytic residues in TmGalA were inferred through structural homology with GH27 members to facilitate design of site-directed mutants. Product analysis confirmed that the wild type (WT) acted with retention of anomeric stereochemistry, analogous to GH27 enzymes. Conserved acidic residues were confirmed through kinetic analysis of D327G and D387G mutant enzymes, azide rescue, and determination of azide rescue products. Mutation of Asp327 to Gly resulted in a mutant that had a 200-800-fold lower catalytic rate on aryl galactosides relative to the WT enzyme. Azide rescue experiments using the D327G enzyme showed a 30-fold higher catalytic rate compared to without azide. Addition of azide to the reaction resulted in formation of azide beta-d-galactopyranoside, confirming Asp327 as the nucleophilic residue. The Asp387Gly mutation was 1500-fold catalytically slower than the WT enzyme on p-nitrophenyl alpha-d-galactopyranoside. Analysis at different pH values produced a bell-shaped curve of the WT enzyme, but D387G exhibited higher activity with increasing pH. Catalyzed reactions with the D387G mutant in the presence of azide resulted in formation of azide alpha-d-galactopryanoside as the product of a retaining mechanism. These results confirm that Asp387 is the acid/base residue of TmGalA. Furthermore, they show that the biochemical characteristics of GH36 TmGalA are closely related to GH27 enzymes, confirming the mechanistic commonality of clan GH-D members.}, number={11}, journal={BIOCHEMISTRY}, author={Comfort, Donald A. and Bobrov, Kirill S. and Ivanen, Dina R. and Shabalin, Konstantin A. and Harris, James M. and Kulminskaya, Anna A. and Brumer, Harry and Kelly, Robert M.}, year={2007}, month={Mar}, pages={3319–3330} }
 @article{suits_sheahan_harris_shafer_degroff_hater_gabr_barlaz_2006, title={Shear strength of degraded reconstituted municipal solid waste}, volume={29}, DOI={10.1520/gtj14089}, abstractNote={Abstract
               Relative changes in Waste shear strength parameters as a function of strain level and stress path are investigated based on the results of 16 direct simple shear (DSS) tests, one direct shear (DS) test with four stages, and three triaxial tests. The magnitudes of shear strength parameters obtained from drained DSS tests and undrained DSS tests with pore water pressure measurement were comparable. This was the case even though the effective stress path in both approaches was different. Data indicated the dependency of the mobilized strength parameters on strain, or deformation level. Generally, stress-deformation response increased monotonically with no well defined peak or ultimate stress levels. The results of the DSS and DS tests show no dependency of the strength parameters on the stress level. Results from DSS and DS indicated a range of effective strength parameters of 9 to 14 kPa for cohesion and 23°–29° for friction angle. Data from the triaxial testing showed dependency of the shear strength parameters on the initial compression stress level. Given the number of potentially confounding issues associated with the measurement of shear strength, it is rather important to also report information on sample collection methods, sample age and chemical composition, sample processing, sample composition, the size of testing equipment and level of strain (instead of ultimate or peak) at which the strength parameters are evaluated.}, number={2}, journal={Geotechnical Testing Journal}, publisher={ASTM International}, author={Suits, L David and Sheahan, TC and Harris, JM and Shafer, AL and DeGroff, W and Hater, GR and Gabr, M and Barlaz, MA}, year={2006}, pages={141–148} }