Optimizing the keratin degradation activity of keratinase via in silico and molecular engineering approaches

Abstract

Keratinases offer a promising green strategy for valorizing recalcitrant keratin waste, yet their practical application is often hindered by low substrate specificity and limited catalytic efficiency. Herein, we report the successful engineering of a highly specific and robust keratinase variant, G209F/G235S, derived from KerBv via a semi-rational design strategy targeting the active pocket. By integrating homology modeling, molecular docking, and evolutionary conservation analysis, key residues within binding pocket were identified and optimized. The engineered mutant exhibited a doubled catalytic activity of 8,540 U/mL and a significantly enhanced keratin-to-casein hydrolytic ratio (K/C) of 1.64. Comprehensive characterization revealed that G209F/G235S possesses superior thermostability with the optimum reaction temperature of the G209F/G235S mutant shifted from 40 °C (WT) to 45 °C, and maintains robust activity across a broad alkaline pH range (7.0–11.0). Molecular dynamics simulations and structural analyses elucidated the underlying mechanisms: the mutations synergistically expanded the active pocket volume from 561 to 689 ų, and optimized electrostatic complementarity for negatively charged keratin. In practical applications, the mutant achieved over 80% degradation of native feather waste within 2 h, outperforming the WT by 50% degradation ratio. This study not only delivers a potent biocatalyst for the efficient upcycling of keratinous by-products but also provides fundamental insights into the structure–function relationships governing keratinase specificity, establishing a robust strategy for the rational design of high-performance industrial enzymes.

Keywords

Keratinase / Substrate specificity / Active pocket engineering / Semi-rational design / Feather degradation / Green biocatalysis

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Funding

the National Natural Science Foundation of China(No. 32301283)

the China Postdoctoral Science Foundation(No. 2025M772526)

the Fundamental Research Funds for the Central Universities(No. JUSRP202501071)

RIGHTS & PERMISSIONS

Jiangnan University