Session: Current Topics in Biological Chemistry:

Organophosphates (OPs) are a class of chemicals which are commonly used as pesticides, but they are highly toxic and pose a threat to human health and the environment. The enzyme phosphotriesterase (PTE) catalyzes the hydrolysis of OPs and is a promising method for detoxification. Fluorination of the enzyme through the incorporation of the non-canonical amino acid para-fluorophenylalanine can be used to improve its stability, but decreases the protein’s solubility, making expression difficult. Here, we use computational design to design PTE variants with high positive or negative surface charges, allowing improved solubility. We express the designed variants in Escherichia coli and purify them using fast protein liquid chromatography. We use dynamic scanning calorimetry and circular dichroism spectroscopy temperature scans to examine the effect of supercharging on the enzyme’s thermostability. In order to determine the effect of supercharging on enzyme kinetics and resilience of the variants to high temperatures, each variant is incubated at 20°C, 30°C, 40°C, and 50°C, then used to catalyze the breakdown of paraoxon, a model OP. The breakdown rates were analyzed to determine the kinetic parameters and temperature resilience for each variant.

Jacob Kronenberg

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