Biocatalysis of chlorophyllase from Phaeodactylum tricornutum in a biphasic organic system

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Abstract
The hydrolytic activity of a partially purified chlorophyllase, obtained from the alga Phaeodactylum tricornutum, was determined in a biphasic organic system using different mixtures of hexane and Tris–HCl buffer solution (20 mmol dm−3, pH 7·5). The most appropriate kinetic parameters were determined for the optimum hydrolytic activity of chlorophyllase including hexane concentration (45%), optimum pH (7·5), time of incubation (7 h), incubation temperature (25°C) and enzyme concentration (10 μg cm−3). The optimum concentrations of magnesium chloride and dithiothreitol, used as activators, were determined to be 4 mmol dm−3 and 5 mmol dm−3, respectively. The results also indicated that diisopropyl fluorophosphate (DIFP) had a mixed competitive–non‐competitive inhibitory effect on chlorophyllase activity. The end‐products of the chlorophyllase hydrolytic reaction were identified and confirmed, using thin‐layer and high‐performance liquid chromatographies, spectrophotometric scanning and Fourier transform infrared spectroscopy.