Li_2026_Enzyme.Microb.Technol_196_110822

Poor thermal stability of Rhizopus oryzae lipase (ROL) has long limited its industrial applicability. In this study, we systematically investigated the underlying mechanism for its inactivation and correspondingly developed an effective stabilization strategy. Kinetic analyses revealed that ROL thermal inactivation process followed a two-step model, demonstrating its inactivation proceeds a distinct intermediate state. Various spectroscopic characterizations further suggested that activity loss was closely associated with the progressive unfolding of the enzyme, as evidenced by the disruption of secondary structures and a pronounced increase in fluorescence intensity resulting from the exposure of hydrophobic clusters. To enhance its thermostability, based on the above-discovered mechanism, a compound stabilizer system was developed and further optimized via a combination of Plackett-Burman design and response surface methodology. The obtained optimal formula comprised of 35.94 % (m/v) glucose, 34.82 % (m/v) sorbitol, and 2.50 mol/L NaCl, which could effectively preserve 97.7 % of the initial activity after 3 h of incubation at 60 degreesC, in contrast to only 29.5 % residual activity of the control. Thus, this study elucidated the underlying thermal inactivation mechanism for ROL and developed a practical and efficient stabilization strategy with potential prospect for industrial application.