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<p>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&#xa0;°C (WT) to 45&#xa0;°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 Å<sup>3</sup>, and optimized electrostatic complementarity for negatively charged keratin. In practical applications, the mutant achieved over 80% degradation of native feather waste within 2&#xa0;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.</p>

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

  • 期刊论文

  • 发表日期：

    2026年4月21日

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  • Yu-Xin Chen,
  • Xiang-Lan Xi,
  • Chang Su,
  • Meng-Ting Tan,
  • Heng Li,
  • Jin-Song Gong,
  • Zheng-Hong Xu,
  • Jin-Song Shi

摘要

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 Å3, 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.