Scholarlink

<p>Chitin, a prevalent biopolymer found in arthropod exoskeletons and fungal cell walls, requires efficient depolymerization to provide bioactive derivatives suitable for agriculture, medicine, and wastewater treatment. Bacterial chitinases, such as the exochitinases produced by <i>Paenibacillus barengoltzii</i>, are potential biocatalysts for chitin recycling; however, they exhibit insufficient activity and stability for industrial use. In this study, Semi-rational engineering and site-directed mutagenesis were employed to enhance catalytic efficiency, resulting in five mutants: M1 (D344Y), M2 (D192Y), M3 (D144Y), M4 (D534G), and M5 (D192A). M3 and M5 significantly increased chitinolytic activity, boosting it by 338% and 276%, respectively, compared to the original enzyme. Further enhancement was achieved by incorporating a chitin-binding domain (CBD), resulting in a six-fold increase in activity with the M35-CBD chimaera. Molecular dynamics (MD) simulations were performed on wild-type, M3, and M5 variations to elucidate the structural basis for these enhancements. M3 (D144Y) exhibited greater structural stability, less RMSD fluctuations, and an improved hydrogen bond network in the substrate-binding region. Notably, enhanced π-stacking interactions facilitated by solvent-exposed aromatic residues, including tryptophan, improved substrate affinity. Unlike the wild-type enzyme, the produced mutants exhibited reduced conformational mobility alongside increased substrate interaction. The results demonstrate the efficacy of targeted mutagenesis and domain engineering in creating stable chitinases for industrial and environmental biotechnology applications.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

Site-directed mutagenesis and semi-rational design to enhance chitinolytic activity of bacterial exochitinase

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年5月21日

查看全文
  • Mati Ullah,
  • Jianda Han,
  • Xiaomei Zhu,
  • Zhongjian Guo,
  • Najumuddin,
  • Muhammad Naeem,
  • Vivian Andoh,
  • Guoqiang Guan,
  • Abebe Bogale,
  • Esmael M. Alyami,
  • Ohoud A. Alghamdi,
  • Turki M. Dawoud,
  • Huayou Chen

摘要

Chitin, a prevalent biopolymer found in arthropod exoskeletons and fungal cell walls, requires efficient depolymerization to provide bioactive derivatives suitable for agriculture, medicine, and wastewater treatment. Bacterial chitinases, such as the exochitinases produced by Paenibacillus barengoltzii, are potential biocatalysts for chitin recycling; however, they exhibit insufficient activity and stability for industrial use. In this study, Semi-rational engineering and site-directed mutagenesis were employed to enhance catalytic efficiency, resulting in five mutants: M1 (D344Y), M2 (D192Y), M3 (D144Y), M4 (D534G), and M5 (D192A). M3 and M5 significantly increased chitinolytic activity, boosting it by 338% and 276%, respectively, compared to the original enzyme. Further enhancement was achieved by incorporating a chitin-binding domain (CBD), resulting in a six-fold increase in activity with the M35-CBD chimaera. Molecular dynamics (MD) simulations were performed on wild-type, M3, and M5 variations to elucidate the structural basis for these enhancements. M3 (D144Y) exhibited greater structural stability, less RMSD fluctuations, and an improved hydrogen bond network in the substrate-binding region. Notably, enhanced π-stacking interactions facilitated by solvent-exposed aromatic residues, including tryptophan, improved substrate affinity. Unlike the wild-type enzyme, the produced mutants exhibited reduced conformational mobility alongside increased substrate interaction. The results demonstrate the efficacy of targeted mutagenesis and domain engineering in creating stable chitinases for industrial and environmental biotechnology applications.