Ionic liquids as promoters of fast lysozyme fibrillation

Abstract

Protein nanofibers are gaining much attention due to their unique mechanical strength, thermal stability and functional properties, thus promoting research on new preparation methodologies. Most fibrillation processes developed so far are time consuming, usually taking from 8 h to 15 h, which represents a major drawback in their use. The use of alternative solvents, such as ionic liquids (ILs) and deep eutectic solvents, as fibrillation agents has been recently reported with considerable reduction in the fibrillation time. This fact encouraged us to study the fibrillation of hen egg white lysozyme (HEWL) in the presence of several ILs from two different families, based on i) imidazolium and ii) cholinium cations, combined with different anions derived from organic acids. The properties of the obtained protein nanofibers were studied using UV–vis and fluorescence spectroscopy, electrophoresis, circular dichroism and electron microscopy. All ILs used were shown to fibrillate HEWL within a few hours, generally achieving over 70–80% of conversion ratios. Typically, worm-like nanofibers were obtained, with 0.3–1 μm of length and 15–40 nm of width, depending on the ILs used. Furthermore, the presence of the acetate anion increases the ability of HEWL to form β-sheet structures. These results show that there is a correlation between the IL chemical structure and the structural features HEWL fibrils, which can be advantageously used in multiple applications in the bionanotechnological field.