Stabilization of lysozyme by benzyl alcohol: surface tension and thermodynamic parameters.

The aim of the study was to understand the effect of benzyl alcohol on biological activity, aggregation behavior, denaturant and heat-induced unfolding of lysozyme. Compatibility studies of lysozyme carried out with a number of anti-microbial preservatives, indicated benzyl alcohol to be the best suppressor of protein aggregation against heat stress. The effect of this preservative was checked at various pH values ranging from 4.0 to 9.0. In spite of reducing the thermal denaturation temperature (T(m)) at all pH values, benzyl alcohol had a stabilizing effect on lysozyme in terms of retaining the biological activity when the enzyme was incubated at 75 degrees C. The reduction in T(m) with increasing benzyl alcohol concentration was correlated with decreasing surface tension of surrounding medium. A detailed thermodynamic study of lysozyme in the presence of benzyl alcohol was carried out at pH 6.2. Change in Gibb's free energy of thermal unfolding at 25 degrees C was found to remain constant in the presence of benzyl alcohol, indicating no interaction of benzyl alcohol with the native protein at room temperature. Both the enthalpy and entropy change at mid point of thermal unfolding were found to increase in the presence of benzyl alcohol indicating the stabilization of partially unfolded state.

Role of benzyl alcohol in the prevention of heat-induced aggregation and inactivation of hen egg white lysozyme.

The aim of the study was to investigate the stability of a model protein, lysozyme, in the presence of the commonly used preservative benzyl alcohol. Techniques including lytic assay, size exclusion chromatography, circular dichroism, differential scanning calorimetry, native polyacrylamide gel electrophoresis and dynamic light scattering were used to study the overall stability of lysozyme in the presence of benzyl alcohol. The stability of lysozyme against thermal stress was higher in the presence of benzyl alcohol. In the presence of 0.5%, 0.9% and 2% v/v benzyl alcohol, the enzyme showed 33%, 42% and 75% residual activity, respectively, when exposed to 75 degrees C for 2 h, as compared to the 22% activity of control sample. A gradual increase in the size of aggregates was observed for the control sample relative to the samples containing benzyl alcohol, as a result of loss of monomer concentration. The effect was found to be concentration-dependent with 2% benzyl alcohol showing maximum prevention of heat-induced unfolding and aggregation. This effect is remarkable since the thermal transition temperature of the enzyme decreases in the presence of benzyl alcohol. Benzyl alcohol favours the thermal denaturation of lysozyme but stabilizes the lysozyme against the heat-induced aggregation.