Stabilization of acid proteases through interaction with polyvalent metal ions: Insights from classical leather tanning theory.

Enhancing the stability of acid proteases is crucial for their long-term storage and diverse industrial applications, particularly in high-temperature or long-duration processes, such as wet blue bating in leather manufacturing. On the basis of classical tanning theory, this study investigated the stabilization effects of tanning metal ions, Cr3+, Al3+, and Zr4+, on pepsin, a model acid protease for wet blue bating. Fluorescence spectroscopy and molecular docking results indicated that Cr3+, Al3+, and Zr4+ interacted with pepsin through static quenching, primarily dominated by hydrogen bonds and van der Waals forces in addition to coordination. Cr3+, Al3+, and Zr4+ did not bind to the active site of pepsin, thereby preserving its catalytic function. Pepsin exhibited enhanced storage stability, heat resistance, and application stability in the presence of these polyvalent metal ions. At molar ratios of pepsin to Cr3+, Al3+, and Zr4+ of 1:28, 1:24, and 1:20, respectively, the residual activities of pepsin were 88-96% after 48 h of incubation at 37 °C, above 92% after 12 h of incubation at 55 °C, and 36-38% after 8 h of reaction at 37 °C, all much higher than those of untreated pepsin. These enhanced stabilities are likely attributable to the formation of stable ground-state complexes between pepsin and metal ions, which reinforces the structural integrity of pepsin. This study not only presents a novel strategy for achieving efficient and stable acid proteases for leather bating but also offers a theoretical foundation for understanding how metal ions stabilize enzymes.