An engineered α-1,3-glucanase-derived probe enables sensitive detection of α-1,3-glucan and reveals its soluble release by Aspergillus

α-1,3-Glucan is a major component of the Aspergillus cell wall. However, unlike β-1,3-glucan and galactomannan, α-1,3-glucan was not previously considered to be released in a soluble form. Therefore, its potential as a diagnostic biomarker has remained largely unexplored. In this study, we engineered α-1,3-glucanase mutants to develop α-1,3-glucan-specific binding proteins and detection systems. Mutants derived from the endo-type α-1,3-glucanases, AGN1 and AglKA, were generated, among which AglKA-CatD-D1090A lacked enzymatic activity but retained strong binding affinity for α-1,3-glucan. Using this probe, we established a split luciferase assay to detect α-1,3-glucan in Aspergillus hyphae and conidia without prior purification. Soluble α-1,3-glucan was substantially released by major Aspergillus species, as indicated by results of a sandwich ELISA-like assay with AglKA mutants. These findings suggest that soluble α-1,3-glucan has potential as a complementary diagnostic biomarker for aspergillosis.