AbstractUsing commercial immobilized glucose isomerase (SWETASE®, Nagase Co.), the effect of substrate protection on enzyme deactivation has been studied in a batch manner. The data analysis was carried out based on Briggs‐Haldane kinetics in which enzyme deactivation accompanying the protection of substrates was also considered. The protection factor was proposed to elucidate the dependence of the degree of substrate protection. The existence of the protection of glucose isomerase by the substrates has been verified experimentally. Also, the enzyme‐substrate complex deactivates with a decay constant which is one‐half that of the free enzyme. Theoretical analysis of enzyme deactivation with substrate protection offers an effective understanding which is essential for enzyme replacement and process optimization.