Combined computational, rational, and empirical design of boiling-resistant keratinase

Engineering of highly thermostable keratinase is of great theoretical interest in understanding protein stability mechanisms and practical significance for processing keratinous wastes such as feathers and wool. The thermostable subtilisin-like protease C2 is the major keratinase secreted by <i>Thermoactinomyces vulgaris</i> CDF but is rapidly inactivated at temperatures above 90°C. Here, we employed various methods to further stabilize protease C2. Four of the 35 selected single-point mutations designed by automated computational tools (PROSS, FireProt, ProteinMPNN, HyperMPNN, and ThermoMPNN) retained higher residual activity (~72%-84%) than protease C2 (~54%) after 1-h incubation at 85°C. The rational design of surface ion pairs and proline substitutions in β-turns generated two single-point variants with increased thermostability. Although three single-point aspartate substitutions appeared to be neutral, they could synergistically or cumulatively improve enzyme stability. The combination of these nine stabilizing mutations yielded the variant SM9 with a half-life of ~4 h at 100°C. The molecular dynamics simulations of protease C2 revealed several relatively flexible regions, including two Ca²⁺-binding sites (Ca1 and Ca2). Empirically modifying the Ca1 site and incorporating an additional two Ca²⁺-binding sites (Ca3 and Ca4) into the flexible regions yielded the variant CM1 with enhanced thermostability. By combining the mutations in SM9 and CM1, the variant CM16 was generated with a half-life of more than 9 h at 100°C. SM9 and CM16 are also highly resistant to high alkalinity, high salinity, urea, sodium dodecyl sulfate (SDS), organic solvents, and reductants, enabling them to efficiently degrade chicken feathers at temperatures near the boiling point.IMPORTANCEThe boiling-resistant enzymes are especially valuable not only for probing the molecular basis that allows proteins to function at the maximum temperature capable of supporting life but also offer the opportunity to greatly expand the enzymatic reaction conditions. Besides exploring naturally occurring boiling-resistant enzymes from hyperthermophiles, artificial engineering of enzymes with boiling resistance remains an important challenge. Our results demonstrate that the thermostability of the subtilisin-like protease C2 with keratinolytic activity can be largely improved by the combined use of automated computational design, structure-based rational design, and empirical engineering. The resulting variants are not only stable and functional at temperatures near or above 100°C but also show improved resistance to polyextreme conditions, providing new clues about the stabilization mechanism of subtilases. Moreover, by virtue of their hyperthermostability, the boiling-resistant variants could be repeatedly used for processing keratin substrates at high temperatures and find practical applications in feed, food, and leather industries.