Our ability to sense small molecules with high specificity, over a broad range of concentrations, is limited and difficult to accomplish in a way that is inexpensive and continuous. The most commercially successful biosensor is the enzyme-based blood glucose electrochemical biosensor, yet for nearly all other biomolecules, detection and monitoring require specialized equipment, trained personnel, and long lead times, and are not amenable to continuous monitoring. Industries in need of enzyme-based small-molecule biosensors, including medical diagnostics, industrial production, environmental monitoring, food safety analysis, and international security, would benefit greatly from the development of new devices capable of measuring biomolecules of interest.
Environmental microbes have been gaining attention because of the vast array of biomolecules that they are capable of sensing and degrading. These microbes do so, in part, through redox enzymes with diverse substrate specificities that represent an immense resource for developing electrochemical biosensors. However, the development of new enzyme biosensors has largely been limited by the lack of a general high-throughput method to identify these redox enzymes, making discovery slow, laborious, and ad hoc.
To address this need, a high-throughput functional screening approach has been developed to isolate microbial oxidase enzymes from complex metagenomic DNA libraries based solely on the enzyme-mediated degradation of any target analyte. The approach can be applied to DNA isolated from any complex microbial sample, including unidentified or unculturable bacteria. In this research, I first describe the development of a general assay to capture the activity of oxidase enzymes expressed in E. coli cells. I then demonstrate how the assay can be used to screen for the nicotine degrading oxidase NicA2 from a genomic DNA library generated from the microbe P. putida. Lastly, I describe the use of this screen to identify a new hydrocortisone-responsive oxidase from a pooled genomic DNA library of eight microbes, representing over 43 Mb of DNA sequence space. This hydrocortisone oxidase represents the first of many new enzymes that can be discovered leveraging our screening platform, which is poised to revolutionize the electrochemical biosensing field and substantially broaden the number of molecules these electrochemical biosensors can detect continuously and quantitatively. / 2023-02-17T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/42066 |
| Date | 18 February 2021 |
| Creators | Ortiz, Luis Angel |
| Contributors | Densmore, Douglas, Galagan, James |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
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