The 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 Mg(2+), the temperature optima for the three soluble enzymes were 85 degrees C (SMGI), 95 degrees to 100 degrees C (TNGI), and >100 degrees 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 Mg(2+). The highest productivities measured were 750 and 760 kg fructose per kilogram SMGI at 60 degrees C and 70 degrees C, respectively. The highest productivity for both TMGI and TNGI in the presence of Mg(2+) occurred at 70 degrees C, pH 7.0, with approximately 230 and 200 kg fructose per kilogram enzyme for TNGI and TMGI, respectively. At 80 degrees C and in the presence of Mg(2+), 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 degrees C and above using TNGI and SMGI under optimal conditions, which included the presence of both Co(2+) and Mg(2+). In the presence of both cations, these enzymes showed the potential to catalyze glucose-to-fructose conversion at 80 degrees C with estimated lifetime productivities on the order of 2000 kg fructose per kilogram enzyme, a value competitive with enzymes currently used at 55 degrees to 65 degrees C, but with the additional advantage of higher fructose concentrations. At 90 degrees 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.