AbstractIrreversible thermoinactivation of immobilized glucose isomerase from Streptomyces olivochromogenes has been mechanistically investigated at the pH‐optimum of enzymatic activity (pH 8.0). Ligands (high fructose corn syrup and the competitive inhibitor xylitol) greatly stabilize the immobilized enzyme at high temperatures. At 90°C in the presence of 2M xylitol, irreversible inactivation of immobilized glucose isomerase is caused by deamidation of its asparagine/glutamine residues. On the basis of the data obtained, it appears that the time‐dependent decay of glucose isomerase activity in industrial bioreactors is brought about by oxidation of the enzyme's cysteine residue and/or heat‐induced deleterious reactions with high fructose corn syrup or its impurities.