Date of Award
8-2026
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Biochemistry and Molecular Biology
Committee Chair/Advisor
Stephen K. Dolan
Committee Member
James C. Morris
Committee Member
Cheryl ingram-Smith
Abstract
Aspergillus fumigatus (Af) is an opportunistic fungal pathogen that thrives in polymicrobial environments such as soil and the lungs of immunocompromised individuals. Survival in these nutrient-limited environments requires efficient nitrogen acquisition, yet the specific enzymes involved and their biological roles remain poorly characterized. Amine oxidases (AOs) catalyze the oxidation of amines, generating aldehydes, ammonia, and hydrogen peroxide, and are thought to contribute to nitrogen metabolism and adaptation in Af. Previous work identified 17 putative AOs, which were narrowed to four candidates that exhibit activity with tested biologically relevant substrates.
This study established a robust system enabling the production, labeling, and functional characterization of these candidate enzymes. The AOs were fused to an ALFA tag and mNeonGreen, enabling detection by fluorescence. To isolate the tagged enzymes, NbALFA, a nanobody that binds tightly to the ALFA tag, was used. Mycelia expressing these constructs were grown at a scale sufficient for downstream analysis, and the capture and labeling strategy successfully prepared enzymes for functional testing. To assess catalytic activity, induced enzymes were incubated with an amine substrate panel, and hydrogen peroxide production was measured as an indicator of catalytic activity.
This approach provides a robust platform for native expression and isolation of candidate AOs for quantitative fluorescence-based assays using horseradish peroxidase (HRP) and Amplex Red to measure hydrogen peroxide production. Through the functional testing of these enzymes, this study establishes a foundation for future investigations into their biochemical roles and contributions to nitrogen metabolism in Af.
Recommended Citation
Husted, Anna, "Biochemical Approaches to Characterize Native Amine Oxidases in Aspergillus fumigatus" (2026). All Theses. 4847.
https://open.clemson.edu/all_theses/4847