Preservation Effect of Vitreous non Reducing Carbohydrates on the Enzymatic Activity, Denaturation Temperature and Retention of Native Structure of Lysozyme

Abstract

In order to develop more efficient methodologies to preserve protein based products at room temperature, a study on the preservation of the enzyme lysozyme in glassy matrices made from three different carbohydrates: trehalose, sucrose and raffinose was carried out. The lysozyme-carbohydrate systems were evaluated structurally by Fourier Transform Infrared (FTIR) spectroscopy, to analyze the intermolecular interactions between carbohydrate and protein. Their thermal stability was characterized using differential thermal analysis (DTA), which allowed the measurement of glass transition temperaturas (T_g) of the vitreous sugars and differential scanning calorimetry (DSC), which was used to measured the T_g of sugars in the sugar enzyme systems as well as the thermal denaturation temperature of lysozyme (T_d). The structural studies revealed that sucrose is the most effective sugar for the preservation of the native conformation of lysozyme during lyophilization. Nevertheless, analysis of enzymatic activity showed, after storing the enzyme at room temperature for more than five weeks, that the highest activity retention was achieved when preserved in the presence of trehalose and raffinose. Freeze-dried lysozyme in the absence of sugars partially lost its native conformation during the lyophilization and lost 20% of its biological activity when stored at room temperature.