Functionally modified chitotriosidase catalytic domain for chitin detection based on split-luciferase complementation

Carbohydr Polym. 2022 Apr 15:282:119125. doi: 10.1016/j.carbpol.2022.119125. Epub 2022 Jan 12.

Abstract

In this study, we applied a luciferase-fragment complementation assay for chitin detection. When luciferase-fragment fused chitin-binding proteins were mixed with chitin, the reconstituted luciferase became active. The recombinant chitin-binding domain (CBD) and a functionally modified catalytic domain (CatD) of human chitotriosidase were employed for this method. We designed the CatD mutant as a chitin-binding protein with diminished chitinolytic activity. The non-wash assay using the CatD mutant had higher sensitivity than CBD for chitin detection and proved to be a structure-specific biosensor for chitin, including crude biomolecules (from fungi, mites, and cockroaches). The CatD mutant recognized a chitin-tetramer as the minimal binding unit and bound chitin at KD 99 nM. Furthermore, a sandwich ELISA using modified CatD showed a low limit of quantification for soluble chitin (13.6 pg/mL). Altogether, our work shows a reliable method for chitin detection using the potential capabilities of CatD.

Keywords: Chit1; Chitin; Chitin binding-module; Chitinase; Chitosan; NanoBiT.

MeSH terms

  • Animals
  • Biosensing Techniques
  • Candida albicans / chemistry
  • Carbohydrates / chemistry
  • Catalytic Domain / genetics
  • Chitin / analysis*
  • Chitin / chemistry
  • Cockroaches / chemistry
  • Dermatophagoides farinae / chemistry
  • Dermatophagoides pteronyssinus / chemistry
  • Enzyme-Linked Immunosorbent Assay
  • Hexosaminidases / chemistry*
  • Hexosaminidases / genetics
  • Luciferases / chemistry
  • Mutation

Substances

  • Carbohydrates
  • Chitin
  • Luciferases
  • Hexosaminidases
  • chitotriosidase