Functionally modified chitotriosidase catalytic domain for chitin detection based on split-luciferase complementation
Functionally modified chitotriosidase catalytic domain for chitin detection based on split-luciferase complementation
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DOI：
10.1016/j.carbpol.2022.119125
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发表时间：
2022-01-18
影响因子：
11.2
通讯作者：
Adachi，Yoshiyuki
中科院分区：
文献类型：
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作者：
Yamanaka，Daisuke;Suzuki，Kento;Adachi，Yoshiyuki
在这项研究中，我们应用了几丁质酶片段互补检测。当将几丁质结合蛋白与几丁质酶片段融合后，重组的荧光素酶具有活性。重组几丁质结合结构域（CBD）和功能修饰的催化结构域（CatD）的人壳三糖苷酶，用于这种方法。我们设计了CatD突变体作为几丁质结合蛋白，具有减弱的几丁质分解活性。使用CatD突变体的非洗涤测定具有比CBD更高的几丁质检测灵敏度，并且被证明是几丁质的结构特异性生物传感器，包括粗生物分子（来自真菌、螨和蟑螂）。CatD突变体识别几丁质四聚体作为最小结合单位，并以KD 99 nM结合几丁质。此外，使用改良的CatD的夹心ELISA显示可溶性几丁质的定量下限（13.6 pg/mL）。总之，我们的工作显示了一个可靠的方法，几丁质检测使用CatD的潜在能力。
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 atKD99 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.