P57: Purification and deactivation study of cutinase from Pseudomonas cepacia NRRL B 2320

An extracellular enzyme cutinase from Pseudomonas cepacia NRRL B2320 was isolated to apparent homogeneity with 89.84 fold purification and 17.56% yield. The enzyme has a molecular mass of 26.5 kDa. The deactivation of the purified protein was studied at different combination of pH (pH 6 to pH 9) and temperature (40ºC to 60 ºC). Increase in entropy and enthalpy values was observed when pH increases from 6 to 7. Beyond pH 7 there was decrease in both the entropy and enthalpy. Positive values of ΔH* would be expected for the breaking of hydrogen bonds as well as for the exposure of hydrophobic groups from the interior of the native, folded protein during the unfolding process. This phenomenon was also supported by fluorescence study during deactivation process. Decrease in intensity and red shift in λ _max was observed with increase in temperature. This is due to the exposure of the tryptophan to an aqueous environment as opposed to a hydrophobic protein interior at higher temperature. Positive value of ΔS* indicates that the protein solution has become more disordered as it deactivated by temperature. This disorderedness of protein was further verified by CD spectroscopy. With the increase in temperature the ellipticity was increases at 208, 220 nm and decreases at 200 nm. This suggests that there is loss of defined secondary structures (α-helix and β-sheet) and formation of more random coil in the protein structure at higher temperature.