Purification and Characterization of Chlorophyllase from Alga (Phaeodactylum tricornutum) by Preparative Isoelectric Focusing

Abstract

Chlorophyllase from a diatom alga (Phaeodactylum tricornutum) was obtained and the partially purified extract has been further purified using preparative isoelectric focusing on a Rotofor cell. Three fractions, FI, FII, and FIII, were separated from the Rotofor cell and salt and ampholytes were removed to give fractions FI', FII', and FIII', respectively. Enzyme fractions FI', FII', and FIII', respectively. Enzyme fractions FI', FII', and FIII' showed specific activities of 15.2×10^-4, 226.7×10^-4, and 33.8×10^-4μmol/mg protein/min, respectively. Most of the enzyme activity (84%) was in fraction FII'. The optimum pH for chlorophyllase activity was 8.0 for FI' and 8.5 for both FII' and FIII'. Apparent K_m values for enzyme fractions FI', FII', and FIII' were 2.1n_M, 2.3n_M, and 2.0n_M, respectively. Enzyme fractions FII' and FIII' showed higher chlorophyllase activity towards the partially purified chlorophyll when it was compared to that with the crude chlorophyll as well as with both chlorophylls a and b. However, the enzyme fraction FI' had higher activity towards the crude chlorophyll when it was compared to that with both chlorophylls a and b, but with a preference for chlorophyll a over chlorophyll b. The inhibitory effect of diisopropyl flurophosphate (DIFP) on chlorophyllase activity demonstrates a noncompetitive inhibitor kinetics with K_i values of 1.29m_M, 2.14m_M, and 0.71m_M for FI'. FII', and FIII', respectively.