Preferential interaction of denaturants with rice bran lipase

The catalytic stability of rice bran lipase has been determined in the presence of three denaturants viz, urea, GuHCl and GuHSCN. The enzyme is completely inactive above 7 M urea, 4 M GuHCl and 2 M GuHSCN concentration. The extent of denaturant interaction has been determined by the partial specific volume measurements of the enzyme. The preferential interaction parameter (xi 3) has values of 0.042, 0.064 and 0.075 g/g, and the denaturation volume changes are -180, -240 and -270 ml/mol in presence of 8 M urea, 6 M GuHC1; and 3 M GuHSCN, respectively. The experimental values of number of denaturant molecules bound (A3) are 0.418, 0.566 and 0.320 g/g and the calculated values are 0.321, 0.511 and 0.632 g/g in presence of 8 M urea, 6 M GuHCl and 3 M GuHSCN, respectively. Fluorescence emission measurements indicated a decrease in the fluorescence emission intensity and a red shift in the emission maximum as the denaturant concentration is increased indicating the gradual exposure of aromatic chromophores. The instability of the enzyme in the presence of these denaturants has been indicated by a decreased value of apparent thermal denaturation temperature (Tm) of the enzyme from a control value of 67 degrees C. The results obtained in the present study explain the extent of inactivation/stability of rice bran lipase in presence of these denaturants at different concentrations.