Changes in protein substrate specificity during inactivation of different Alcalase formulations depends on inactivation conditions

Free and carrageenan immobilized Alcalase preparations were incubated at 70ºC and pHs 5, 7, and 10, and their activities were monitored with the synthetic substrate Boc-L-Ala-4-nitrophenyl ester (NPA), casein, bovine serum albumin and hemoglobin. The inactivation profiles were fully different when followed using the different substrates. Stability was much lower when using NPA compared to the results obtained when using the substrate proteins. A great diversity could also be observed using the different protein substrates, however, the highest stability for the free and immobilized Alcalase was observed using different substrate proteins depending on the inactivation conditions, thus affecting the stabilization factors achieved by the immobilization (e.g., stabilization caused by the immobilization was negligible at pH 10 using HG, but very relevant at pH 5 using NPA). This was explained because the new enzyme conformations generated during inactivation, which depend on the inactivation conditions and enzyme formulations, have new protein substrate specificities.