A Novel Method of Extracting Horseradish Peroxidase from Horseradish Roots Using Ethanol Precipitation

Overview

This project sought to explore accessible means for extracting horseradish peroxidase (HRP), an enzyme commonly utilized in chemical and biological experiments. While purified HRP can be easily obtained commercially, accessibility is compromised by its high cost, which limits the use of the enzyme for educational purposes. In addition, some established protocols for the extraction of enzymes require expensive equipment, such as an ultrasonicator probe and a hydrophobic interaction chromatography apparatus, tools unavailable in a high school setting. To overcome the restriction associated with the inaccessible equipment, single-factor experiments were carried out to determine whether HRP extraction was possible utilizing solely accessible high school resources. It was hypothesized that the higher the concentration of ethanol used during precipitation, the greater the activity of the purified enzyme. The findings obtained support the hypothesis, as higher concentrations of ethanol allowed for faster rates of colorimetric reactions. Since there was no functioning spectrophotometer, the quantifiable measurements of absorbance were done through Python image analysis that traced changes in RGB over time and translated them to Delta E for color change visualizations. Camera-based analysis can possibly distort color and decrease the accuracy of the measurement, potentially causing slight inconsistencies observed. It was found that 80% ethanol concentration is optimal for precipitation of HRP. The trials demonstrate that this accessible extraction method is both reproducible and highly cost-effective, reducing the expenses of a standard equipment-intensive workflow from $10,310 to approximately $200. This 98% cost reduction emphasizes the method's utility, proving that functional HRP can be extracted in limited environments without compromising experimental integrity. By providing a viable alternative to expensive, commercially prepared enzymes, this approach significantly expands access to biochemical experiments for educational settings and resource-poor laboratories.

Competition history

• CSEF 2026 Biochemistry/ Molecular Biology (Senior Division) · Entry S-04-45

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