Isolation, purification and determinatination of some biochemichal properties of pectin methylesterase from black carrot

This research was undertaken to determine some of the biochemical properties (substate specificity, optimum pH, optimum temperature, effect of salts, thermal stability and inactivation) of pectin methylesterase isolated from black carrot and partially purified by 80% ammonium sulphate precipitation followed by dialysis.Km and Vmax values of the enzyme using apple pectin as substrate were 2.14 mg/ml (r2=0.988) and 3.75 unit/ml respectively. The optimum pH for PME activity was found to be 7.5 and the enzyme showed high activity over a broad pH range of 6.0-8.5. The optimum temperature for PME activity was 55°C, after which enzyme activity dropped sharply. Enzyme activity was more than 80% between 50-60°C. The enzyme was very stabile between 30-50°C and the stability declined after 50°C. According to thermal inactivation studies kD values increased as the temperature incresed whereas half-life and D values decreased. Energy of activation (Ea) and Z values were found to be 196.8 kJmol-1 (r2=0.9957) and 2.16ºC (r2=0.9945), respectively. As the Z value indicates the enzyme is not very thermostabile.