Modifying the Substrate Specificity of Keratinase for Industrial Dehairing to Replace Lime-Sulfide

Keratinase has shown potential as an ecofriendly alternative to toxic lime-sulfide for industrial dehairing. However, low substrate specificity usually causes collagen damage. Here, we developed a variant of keratinase KerZ1 with low collagenase activity by modifying the substrate binding pocket. First, sequence alignment identified several targets for distinguishing substrate selectivity. Although the keratinase activity decreased by 41.16 and 22.99%, the collagenase activity of variants Y208V and L216S decreased by 70.91 and 49.51%. On the other hand, molecular docking of KerZ1 with the characteristic peptide FALGPA also revealed that the keratinase activity of variant S100D increased by 5.45%, and the collagenase activity decreased by 25.90%. Next, the keratinase activity of the double mutagenesis variant S100D/Y208V was only 0.04% lower than that of KerZ1, while collagenase activity greatly reduced by 60%. Moreover, the catalytic efficiency ( K cat / K m ) of the variant S100D/Y208V on collagen decreased by 79.60%. For dehairing, S100D/Y208V not only significantly reduced the damage to cowhide and sheepskin collagen but also maintained the same dehairing ability as KerZ1. In fed-batch fermentation, the keratinase activity of S100D/Y208V increased by 9.28 times. This study suggests that improving the substrate specificity will promote keratinase to replace toxic lime-sulfide for industrial dehairing.