Date of Graduation

5-2026

Document Type

Thesis

Degree Name

Bachelor of Science in Chemistry

Degree Level

Undergraduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Susanne Striegler

Committee Member

Nan Zheng

Second Committee Member

Josh Sakon

Third Committee Member

Kate Chapman

Abstract

Raffinose family oligosaccharides are important transport carbohydrates in many cucurbit species that undergo rapid hydrolysis in sink tissues during fruit development. α-Galactosidase (Enzyme Commission 3.2.1.22) catalyzes the cleavage of terminal α-D-galactosyl residues from oligosaccharides, linking plant carbon allocation to applications in food and biotechnology. α-Galactosidase has applications spanning biomedicine and food processing, yet commercial supplies are costly and inconsistent. This study developed an activity guided workflow to isolate and enrich α-galactosidase activity from readily available cantaloupe (Cucumis melo) fruit tissue. Ammonium sulfate precipitation provided stronger enrichment when compared to polyethylene glycol precipitation and produced a concentrated preparation suitable for downstream processing. Ultrafiltration localized measurable activity to the higher mass fraction, which is consistent with the α-galactosidase existing as a large enzyme or oligomeric form with a molecular weight above 50 kDa. Gel permeation chromatography displayed multiple high molecular mass species in the enriched fractions, supporting the presence of more than one protein component in the preparation. These results establish a reproducible enrichment strategy for functional cantaloupe α-galactosidase and for downstream separations, isoform resolution, and kinetic and inhibition studies.

Keywords

α-galactosidase; Cucumis melo; enzyme purification; ammonium sulfate precipitation; raffinose family oligosaccharides; ultrafiltration

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