Thermodynamic and Physical Analysis of the Multi-Stage Thermal Inactivation Processes of Kluyveromyces marxianus Inulinase

Abstract

The thermal inactivation behavior of free inulinase derived from yeast Kluyveromyces marxianus was examined using differential thermal analysis (DTA) over the temperature range of 30-80 °C. The thermograms revealed two distinct endothermic transitions at 54 °C and 65 °C, indicating a multistage denaturation process. The initial transition corresponds to reversible local conformational rearrangements, whereas the subsequent transition is associated with irreversible unfolding and loss of catalytic activity. The observed thermal behavior is attributed to an increase in the heat capacity of the denatured protein, resulting from enhanced exposure of hydrophobic side chains to the solvent. At the optimal temperature, the enzyme adopts a conformation that is both energetically and sterically favorable for efficient inulin hydrolysis. These findings provide insight into the thermodynamic nature of enzyme stability and inactivation, which is relevant for optimizing enzymatic processes and guiding strategies for enzyme reuse or immobilization.