Water soluble nanotubes, that have inner diameters comparable to biomacromolecules, can serve as nanocontainers that can protect proteins from thermal and chemical stressors. The confinement of proteins within the nanotubes can restrict the unfolding of proteins and therefore have a stabilizing effect. The application of these nanocontainers for biomolecules include drug delivery and vaccinations in the absence of refrigeration. The ability of self-assembling lithocholic acid (LCA) nanotubes to encapsulate enzymes, and protect these enzymes from thermal and chemical stresses was investigated in this study. LCA nanotubes were self-assembled by dissolving LCA in a basic sodium hydroxide solution (pH 13), then adding hydrochloric acid solutions (1 M or 0.1 M) drop-wise to lower the pH to 8. Previous studies have shown that the LCA nanotubes self-assembled in this manner, have an inner diameter of 20-40 nm. The enzyme, α-chymotrypsin from bovine pancreas, was encapsulated in the self-assembled LCA nanotubes by introducing the enzyme to a refrigerated solution of LCA nanotubes and then allowing a minimum of five days for the enzyme to diffuse into the nanotubes. Free enzyme was removed by filtering the nanotube suspensions, then re-suspending the nanotubes in a pH 8 solution. The total chymotrypsin encapsulated within the nanotubes was then determined by completing a Pierce BCA protein assay. The concentration of encapsulated chymotrypsin was determined to be 150 µg/mL. A fluorometric Proteasome 20S assay was used to determine the kinetic activity of the chymotrypsin enzyme. Specifically, chymotrypsin cleaves the LLVY-R110 fluorogenic substrate in the Proteasome 20S assay to produce a fluorescent product (λ<sub>ex</sub> = 480-500 nm, λ<sub>em</sub> = 520-530 nm). The rate of formation of the fluorescent product was monitored over a time to determine the enzymatic activity. This study will discuss the development of the experimental methods used to investigate the activity of chymotrypsin encapsulated in LCA nanotubes, and also to present results of studies investigating the ability of LCA nanotubes to protect encapsulated chymotrypsin from thermal and chemical stressors.

Catalytic activity and thermal stability of chymotrypsin encapsulated in self-assembled lithocholic acid nanotubes

253rd National Meeting (2017)

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