Re-engineering lysozyme solubility and activity through surfactant complexation

Abstract

Hydrophobic ion-pairing is an established solubility engineering technique that uses amphiphilic surfactants to modulate drug lipophilicity and facilitate encapsulation in polymeric and lipid-based drug delivery systems. For proteins, surfactant complexation can also lead to unfolding processes and loss in bioactivity. In this study, we investigated the impact of two surfactants, sodium dodecyl sulphate (SDS) and dioctyl sulfosuccinate (DOSS) on lysozyme's solubility, activity, and structure. SDS and DOSS were combined with lysozyme at increasing charge ratios (4 : 1, 2 : 1, 1 : 1, 1 : 2 and 1 : 4) via hydrophobic ion pairing at pH 4.5. Maximum complexation efficiency at the 1 : 1 charge ratio was confirmed by protein quantitation assays and zeta potential measurements, showing a near neutral surface charge. Lysozyme lipophilicity was successfully increased, with log D n-octanol/PBS values up to 2.5 with SDS and 1.8 with DOSS. Bioactivity assays assessing lysis of M. lysodeikticus cell walls showed up to a 2-fold increase in lysozyme's catalytic ability upon complexation with SDS at ratios less than stoichiometric, suggesting favourable mechanisms of stabilisation. Secondary structural analysis using Fourier-transform infrared spectroscopy indicated that lysozyme underwent a partial unfolding process upon complexation with low SDS concentrations. Molecular dynamic simulations further confirmed that at these low concentrations, a positive conformation was obtained with the active site residue Glu 35 more solvent-exposed. Combined, this suggested that sub-stoichiometric SDS altered the active site's secondary structure through increased backbone flexibility, leading to higher substrate accessibility. For DOSS, low surfactant concentrations retained lysozyme's native function and structure while still increasing the protein's lipophilic character. Our research findings demonstrate that modulation of protein activity can be related to surfactant chemistry and that controlled ion-pairing can lead to re-engineering of lysozyme solubility, activity, and structure. This has significant implications for advanced protein applications in healthcare, particularly towards the development of formulation strategies for oral biotherapeutics.