protease K, substrate binding pocket, catalytic efficient, wool scale layer, biodegradation, Fermentation industries. Beverages. Alcohol, TP500-660

The outermost surface of wool is covered by a scale layer, posing challenges to some steps of fabric processing. This layer, primarily composed of keratin, resists degradation by conventional proteases due to its high disulfide bond content. Protease K, an extracellular serine endo-proteinase derived from Tritirachium album Limber (tPRK), is known for its ability to digest native keratin. However, its limited activity against keratin has restricted its application in wool scale layer treatment. In this study, the substrate-binding pocket of tPRK was engineered, yielding the mutant N162A, which demonstrated an 84% increase in catalytic activity toward keratin. Additionally, the catalytic efficiency (kcat/Km) of N162A on keratin improved by 44.52%. Structural analysis indicated that modifications in the substrate-binding pocket reduced steric hindrance during substrate entry while enhancing substrate binding. Additionally, 3.3 mg/mL of amino acids were released within 6 h, which were catalyzed by N162A, with a 61% increase compared to the native tPRK. Moreover, the N162A variant effectively reduced the scale layer thickness without compromising the tensile strength of the wool, maintaining its mechanical properties. The findings provide a sustainable strategy for the wool industry while broadening the scope of biotechnological applications in the textile sector.