キムラ　マサヒロ   木村　将大    所属   応用生物学部 応用生物学科    職種   助教
言語種別	英語
発行・発表の年月	2022/04
形態種別	学術論文
査読	査読あり
標題	Functionally modified chitotriosidase catalytic domain for chitin detection based on split-luciferase complementation.
執筆形態	共著
掲載誌名	Carbohydr Polym
掲載区分	国外
巻・号・頁	282,pp.119125-119125
著者・共著者	Yamanaka D., Suzuki K., Kimura M, Oyama F., Adachi Y.
概要	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.
外部リンクURL	https://www.sciencedirect.com/science/article/pii/S0144861722000297?via%3Dihub