Kinetic stabilization of lysozyme upon interactions with β-cyclodextrin through partial unfolding

Abstract

Protein aggregation and denaturation are two major limitations in bioprocess engineering and protein processing. In the present study, we investigated the effects of β-cyclodextrin, as an artificial chaperone, on the structure and function of lysozyme using UV-Vis, fluorescence spectrophotometry, isothermal titration calorimetry (ITC) and theoretical approaches of docking. Lysozyme was entirely aggregated in a solution containing 0.1% lysozyme (w/v), 150 mM sodium phosphate buffer (pH 7.2), and 10 mM dithiotreitol (DTT). The absorption changes were monitored at 37 °C for 50 min using a UV-Vis spectrophotometer at 360 nm. The residual lysozyme activity was determined using chitosan, a polysaccharide, whose structure is similar to a bacterial cell wall, as a substrate. The effect of β-cyclodextrin on lysozyme activity was determined in the presence and absence of DTT. Our findings indicated that β-cyclodextrin increased the enzyme activity and its kinetics stabilization by binding to Trps 62 and 63, which are embedded in the core of the enzyme inducing its disaggregation. These occurred through the disruption of hydrophobic interactions as demonstrated by ANS fluorescence spectrophotometry. Furthermore, ITC analysis indicated that the binding of β- cyclodextrin to lysozyme was an endothermic reaction and reduced thermodynamic stability by partial unfolding of the enzyme. Thus, the interaction of β-cyclodextrin with lysozyme reduces themodynamic stability by inducing partial unfolding of the enzyme.