Publication Date

Spring 4-27-2024

School

School of Health Sciences

Major

Chemistry

Keywords

peroxidase, butternut squash, purification, thermal stability, biochemical analysis

Disciplines

Biochemistry | Chemistry | Enzymes and Coenzymes | Laboratory and Basic Science Research | Other Chemistry

Abstract

Peroxidases are enzymes that catalyze the reduction of hydrogen peroxide to water while oxidizing organic substrates and are valuable in spheres like industrial and medical applications and histochemistry. Limitations still exist in the use of the well-studied horseradish peroxidase for certain activities due to limitations like poor thermal stability, thus the search for novel peroxidases that can overcome these limitations is an active area of research. Butternut squash peroxidase (Cucurbita moschata) (BSP) shows promise due to significant activity being found in the skin and apparent enhanced thermal stability, but an efficient purification scheme for it is lacking, as well as a detailed characterization of its thermal stability. The preliminary steps of a purification scheme for BSP were developed through methods like “salting out” precipitation techniques, affinity chromatography, and size exclusion chromatography. The efficacy of these techniques was evaluated through two assays. A kinetic assay served to quantify units of activity. A Bradford assay method used an external standard calibration curve to calculate the protein content of samples. The combination of these assays resulted in specific activity measurements that were used to assess the purity of the sample and show the effectiveness of the purification techniques. The thermal stability of BSP was then analyzed and compared to that of HRP, showing that BSP has a higher thermal stability than HRP by 10-20 °C.

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