Thermal inactivation studies of protein: a step toward their biostabilization

In this study the thermal inactivation of pure Yeast Alcohol Dehydrogenase was carried out both in presence and absence of carriers using the activity assay method. The enzyme activity was studied in a UV Spectrophotometer at 340 nm at room temperature and 8.8 pH using NAD as a co-enzyme. The objective of the study was to find out the change in activity or rather the increase in stability of the enzyme in the presence of salts and sugars of known concentrations and subsequent heat treatment in a constant temperature water bath at various temperatures. The first step of the experiment was to choose appropriate inorganic salts and sugars which can provide protection to the enzymes when they are subjected to heat treatment. The next step was to measure the rate of change of absorbance of UV light in a UV Spectrophotometer when only the enzyme (i.e. pure yeast ADH with no other carrier such as salts and sugars) reacts with ethanol and NAD in the assay mixture. The enzyme activity (E) was calculated at various time intervals between 0-60 mins for different values of slopes obtained in the Absorbance-vs-Time curve. A graph of ln(E/Eo)-vs-Time was plotted and nature of the curve was studied and reported. In the next step suitable carriers (such as D-Mannitol, Ammonium Sulphate, Sucrose, etc) were added to the enzyme and similar procedures were followed to obtain the ln(E/Eo) graph. The plot of ln(E/Eo)-vs-Time showed a nonlinear biphasic behavior of the enzyme when subjected to thermal inactivation. This nonlinear behaviour could be due to the formation of enzyme groups with different thermal stabilities or the presence of stable/ labile isoenzymes. It was also observed that with the increase in the percentage of the excipients in the final solution better stability was achieved, even at higher temperatures. It was also observed that when salts (Sodium Sulphate & Ammonium Sulphate ) were used as excipients higher enzyme stability was achieved in comparison to sugars (Sucrose & Trehalose)