@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{kaczowka_madding_epting_kelly_cianciolo_pizzo_2008, title={Probing the stability of native and activated forms of alpha(2)-macroglobulin}, volume={42}, ISSN={["0141-8130"]}, DOI={10.1016/j.ijbiomac.2007.09.019}, abstractNote={alpha2-Macroglobulin (alpha2M) is a 718 kDa homotetrameric proteinase inhibitor which undergoes a large conformational change upon activation. This conformational change can occur either by proteolytic attack on an approximately 40 amino acid stretch, the bait region, which results in the rupture of the four thioester bonds in alpha2M, or by direct nucleophilic attack on these thioesters by primary amines. Amine activation circumvents both bait region cleavage and protein incorporation, which occurs by proteolytic activation. These different activation methods allow for examination of the roles bait region cleavage and thioester rupture play in alpha2M stability. Differential scanning calorimetry and urea gel electrophoresis demonstrate that both bait region cleavage and covalent incorporation of protein ligands in the thioester pocket play critical roles in the stability of alpha2M complexes.}, number={1}, journal={INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES}, author={Kaczowka, Steven J. and Madding, Lara S. and Epting, Kevin L. and Kelly, Robert M. and Cianciolo, George J. and Pizzo, Salvatore V.}, year={2008}, month={Jan}, pages={62–67} }
 @article{madding_michel_shockley_conners_epting_johnson_kelly_2007, title={Role of the beta 1 subunit in the function and stability of the 20S proteasome in the hyperthermophilic archaeon Pyrococcus furiosus}, volume={189}, ISSN={["0021-9193"]}, DOI={10.1128/JB.01382-06}, abstractNote={ABSTRACTThe hyperthermophilic archaeonPyrococcus furiosusgenome encodes three proteasome component proteins: one α protein (PF1571) and two β proteins (β1-PF1404 and β2-PF0159), as well as an ATPase (PF0115), referred to as proteasome-activating nucleotidase. Transcriptional analysis of theP. furiosusdynamic heat shock response (shift from 90 to 105°C) showed that the β1 gene was up-regulated over twofold within 5 minutes, suggesting a specific role during thermal stress. Consistent with transcriptional data, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that incorporation of the β1 protein relative to β2 into the 20S proteasome (core particle [CP]) increased with increasing temperature for both native and recombinant versions. For the recombinant enzyme, the β2/β1 ratio varied linearly with temperature from 3.8, when assembled at 80°C, to 0.9 at 105°C. The recombinant α+β1+β2 CP assembled at 105°C was more thermostable than either the α+β1+β2 version assembled at 90°C or the α+β2 version assembled at either 90°C or 105°C, based on melting temperature and the biocatalytic inactivation rate at 115°C. The recombinant CP assembled at 105°C was also found to have different catalytic rates and specificity for peptide hydrolysis, compared to the 90°C assembly (measured at 95°C). Combination of the α and β1 proteins neither yielded a large proteasome complex nor demonstrated any significant activity. These results indicate that the β1 subunit in theP. furiosus20S proteasome plays a thermostabilizing role and influences biocatalytic properties, suggesting that β subunit composition is a factor in archaeal proteasome function during thermal stress, when polypeptide turnover is essential to cell survival.}, number={2}, journal={JOURNAL OF BACTERIOLOGY}, author={Madding, Lara S. and Michel, Joshua K. and Shockley, Keith R. and Conners, Shannon B. and Epting, Kevin L. and Johnson, Matthew R. and Kelly, Robert M.}, year={2007}, month={Jan}, pages={583–590} }
 @article{mcguffey_epting_kelly_foegeding_2005, title={Denaturation and aggregation of three alpha-lactalbumin preparations at neutral pH}, volume={53}, ISSN={["1520-5118"]}, DOI={10.1021/jf048863p}, abstractNote={The denaturation and aggregation of reagent-grade (Sigmaalpha-La), ion-exchange chromatography purified (IEXalpha-La), and a commercial-grade (Calpha-La) alpha-lactalbumin were studied with differential scanning calorimetry (DSC), polyacrylamide gel electrophoresis, and turbidity measurement. All three preparations had similar thermal denaturation temperatures with an average of 63.7 degrees C. Heating pure preparations of alpha-lactalbumin produced three non-native monomer species and three distinct dimer species. This phenomenon was not observed in Calpha-La. Turbidity development at 95 degrees C (tau95 degrees C) indicated that pure preparations rapidly aggregate at pH 7.0, and evidence suggests that hydrophobic interactions drove this phenomenon. The Calpha-La required 4 times the phosphate or excess Ca2+ concentrations to develop a similar tau95 degrees C to the pure preparations and displayed a complex pH-dependent tau95 degrees C behavior. Turbidity development dramatically decreased when the heating temperature was below 95 degrees C. A mechanism is provided, and the interrelationship between specific electrostatic interactions and hydrophobic attraction, in relation to the formation of disulfide-bonded products, is discussed.}, number={8}, journal={JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY}, author={McGuffey, MK and Epting, KL and Kelly, RM and Foegeding, EA}, year={2005}, month={Apr}, pages={3182–3190} }
 @article{epting_vieille_zeikus_kelly_kelly_zeikus_vieille_2005, title={Influence of divalent cations on the structural thermostability and thermal inactivation kinetics of class II xylose isomerases}, volume={272}, ISSN={["1742-4658"]}, DOI={10.1111/j.1742-4658.2005.04577.x}, abstractNote={The effects of divalent metal cations on structural thermostability and the inactivation kinetics of homologous class II d‐xylose isomerases (XI; EC 5.3.1.5) from mesophilic (Escherichia coli and Bacillus licheniformis), thermophilic (Thermoanaerobacterium thermosulfurigenes), and hyperthermophilic (Thermotoga neapolitana) bacteria were examined. Unlike the three less thermophilic XIs that were substantially structurally stabilized in the presence of Co2+ or Mn2+ (and Mg2+ to a lesser extent), the melting temperature [(Tm) ≈100 °C] of T. neapolitana XI (TNXI) varied little in the presence or absence of a single type of metal. In the presence of any two of these metals, TNXI exhibited a second melting transition between 110 °C and 114 °C. TNXI kinetic inactivation, which was non‐first order, could be modeled as a two‐step sequential process. TNXI inactivation in the presence of 5 mm metal at 99–100 °C was slowest in the presence of Mn2+[half‐life (t1/2) of 84 min], compared to Co2+ (t1/2 of 14 min) and Mg2+ (t1/2 of 2 min). While adding Co2+ to Mg2+ increased TNXI's t1/2 at 99–100 °C from 2 to 7.5 min, TNXI showed no significant activity at temperatures above the first melting transition. The results reported here suggest that, unlike the other class II XIs examined, single metals are required for TNXI activity, but are not essential for its structural thermostability. The structural form corresponding to the second melting transition of TNXI in the presence of two metals is not known, but likely results from cooperative interactions between dissimilar metals in the two metal binding sites.}, number={6}, journal={FEBS JOURNAL}, author={Epting, KL and Vieille, C and Zeikus, JG and Kelly, RM and Kelly, RM and Zeikus, JG and Vieille, C}, year={2005}, month={Mar}, pages={1454–1464} }
 @article{bandlish_hess_epting_vieille_kelly_2002, title={Glucose-to-fructose conversion at high temperatures with xylose (glucose) isomerases from Streptomyces murinus and two hyperthermophilic Thermotoga species}, volume={80}, ISSN={["0006-3592"]}, DOI={10.1002/bit.10362}, abstractNote={AbstractThe conversion of glucose to fructose at elevated temperatures, as catalyzed by soluble and immobilized xylose (glucose) isomerases from the hyperthermophiles Thermotoga maritima (TMGI) and Thermotoga neapolitana 5068 (TNGI) and from the mesophile Streptomyces murinus (SMGI), was examined. At pH 7.0 in the presence of Mg2+, the temperature optima for the three soluble enzymes were 85°C (SMGI), 95° to 100°C (TNGI), and >100°C (TMGI). Under certain conditions, soluble forms of the three enzymes exhibited an unusual, multiphasic inactivation behavior in which the decay rate slowed considerably after an initial rapid decline. However, the inactivation of the enzymes covalently immobilized to glass beads, monophasic in most cases, was characterized by a first‐order decay rate intermediate between those of the initial rapid and slower phases for the soluble enzymes. Enzyme productivities for the three immobilized GIs were determined experimentally in the presence of Mg2+. The highest productivities measured were 750 and 760 kg fructose per kilogram SMGI at 60°C and 70°C, respectively. The highest productivity for both TMGI and TNGI in the presence of Mg2+ occurred at 70°C, pH 7.0, with approximately 230 and 200 kg fructose per kilogram enzyme for TNGI and TMGI, respectively. At 80°C and in the presence of Mg2+, productivities for the three enzymes ranged from 31 to 273. A simple mathematical model, which accounted for thermal effects on kinetics, glucose–fructose equilibrium, and enzyme inactivation, was used to examine the potential for high‐fructose corn syrup (HFCS) production at 80°C and above using TNGI and SMGI under optimal conditions, which included the presence of both Co2+ and Mg2+. In the presence of both cations, these enzymes showed the potential to catalyze glucose‐to‐fructose conversion at 80°C with estimated lifetime productivities on the order of 2000 kg fructose per kilogram enzyme, a value competitive with enzymes currently used at 55° to 65°C, but with the additional advantage of higher fructose concentrations. At 90°C, the estimated productivity for SMGI dropped to 200, whereas, for TNGI, lifetime productivities on the order of 1000 were estimated. Assuming that the most favorable biocatalytic and thermostability features of these enzymes can be captured in immobilized form and the chemical lability of substrates and products can be minimized, HFCS production at high temperatures could be used to achieve higher fructose concentrations as well as create alternative processing strategies. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 185–194, 2002.}, number={2}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Bandlish, RK and Hess, JM and Epting, KL and Vieille, C and Kelly, RM}, year={2002}, month={Oct}, pages={185–194} }