Engineering flexible loops to enhance thermal stability of keratinase for efficient keratin degradation.

Keratinase-catalyzed degradation of keratin waste has been shown to be a promising recycling method. Although the recombinant KerZ1 derived from Bacillus subtilis has shown the highest activity among the keratinases reported so far, the low thermal stability caused by the unstable flexible loops limited its keratin-degrading ability. To this end, the flexible loops of KerZ1 were engineered to be more hydrophobic and rigid through B-factor calculations, molecular dynamics simulations, and β-turn redesign. We developed several highly thermostable keratinase variants and showed enhanced keratin degradation activity. In particular, the loop regions of the variants KerZ1, KerZ1 and KerZ1 were designed to be more stable, with T values increased by 8 °C, 6 °C and 5 °C, and corresponding t increased by 2.3, 3.3 and 5.0 times. The keratin degradation activity of the variant KerZ1 at 60 °C was enhanced by 46 % compared with KerZ1. The strategy of this research and the obtained keratinase variants will be a significant improvement in the complete degradation of keratin.