Joanna Zajda

Protective encapsulation of hydrolase enzymes substrates

Joanna Zajda, WTU ,
Supervisors: prof. Elżbieta Malinowska (WTU), dr. hab. Agata Michalska-Maksymiuk (UW)

Hydrolases substrates intended for fluorimetric determination of enzymes activity are due to their nature highly sensitive to e.g. storage or contact conditions. Upon contact with e.g. alkaline pH spontaneous hydrolysis occurs resulting in formation of fluorescent products similarly as in the case of enzymatic reaction. This process can result in erroneous result of analysis, apart from unwanted losses of expensive substrate. Therefore, the aim of this work 6 was to explore the possibilities of encapsulation of the fragile substrate within the polymeric material to prevent unwanted, non-enzymatic, hydrolysis.
As a model material relatively lipophilic poly(n-butyl acrylate) microspheres were used. The applicability of this approach was verified using enzyme lipase from porcine pancreas and fluorogenic substrate 4-methylumbelliferyl oleate (4-MUO). Poly(n-butyl acrylate) microspheres were obtained in course of emulsion polymerization initiated by UV irradiation. Thus the 4-MUO was incorporated, post polymerization of microspheres by physical adsorption.
It was found that substrate encapsulated in polyacrylate microspheres results in prevention of unwanted hydrolysis even during long contact with alkaline solution. On the contrary, for substrate directly added to the above mentioned solution, spontaneous formation of fluorescent product was observed.
Despite encapsulation the substrate is accessible for enzymatic hydrolysis, resulting in formation of highly fluorescent 4-methylumbelliferone (4-MU) upon contact with lipase in solution. The increase of the activity of the enzyme in solution was leading to increase of fluorescence intensity at 450 nm, i.e. at wavelength characteristic for 4-MU.
Kinetic measurements suggest that the enzymatic reaction occurs at the surface or in the diffusion layer next to the microsphere. Moreover, introduction of either Ca²⁺ ions or bovine serum albumin (BSA) contributes to increase in fluorescence intensity. Zeta potentials data suggest that this effect is caused by absorption of proteins on the microspheres surface; this process is resulting in formation of more enzyme friendly microenvironment and ultimately leads to better sensitivity of the determination.

Chairman - prof. Rafał Siciński

Discussion