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Purification and characterization of chlorophyllase from algaPhaeodactylum tricornutum by preparative native electrophoresis

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Abstract

The partially purified chlorophyllase, obtained from the algaPhaeodactylum tricornutum, was further purified by preparative native gel electrophoresis. The purification procedure provided the recovery of large amounts of a single purified chlorophyllase fraction. However, the electrophoretic analyses of the purified enzymatic fraction under denaturing conditions demonstrated the presence of two bands with mol wt of 43 ±3 and 46 ±3 kDa. The purification procedure resulted in 2-and 195-fold increases in chlorophyllase activity compared to that of the partially purified and crude enzymatic extracts, respectively. The optimum pH for chlorophyllase hydrolytic activity was found to be 8.0. The optimum incubation time and temperature for the hydrolytic activity of the purified chlorophyllase were found to be 2 h and 31°C, respectively. The optimum concentrations of magnesium chloride and dithiothreitol, used as activators, were 4 and 5 mM, respectively. The addition of individual plant membrane lipids, including phosphatidylcholine, phosphatidylglycerol, and β-carotene, to the reaction media increased the enzyme activity markedly. The purified enzyme fraction displayed preferential specificity toward selective substrates with an order of activity as follows: purified chlorophyllb > purified chlorophylla > partially purified chlorophyll > crude chlorophyll. Diisopropyl fluorophosphate and phytol, respectively, showed noncompetitive and competitive inhibitory effects on chlorophyllase activity with Ki, values of 0.78 mM and 3.75μM, respectively.

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Authors and Affiliations

  1. Department of Food Science and Agricultural Chemistry, McGill University, 21, 111 Lakeshore, H9X 3V9, Ste Anne de Bellevue, Qu’ebec, Canada

    A. Khalyfa, S. Kermasha & M. Goetghebeur

  2. Centre Oc’eanologie de Rimouski, INRSOc’eanologie, Rimouski, Qu’ebec, Canada

    P. Marsot

Authors
  1. A. Khalyfa
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  2. S. Kermasha
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  3. P. Marsot
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  4. M. Goetghebeur
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Khalyfa, A., Kermasha, S., Marsot, P. et al. Purification and characterization of chlorophyllase from algaPhaeodactylum tricornutum by preparative native electrophoresis. Appl Biochem Biotechnol 53, 11–27 (1995). https://doi.org/10.1007/BF02783478

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  • DOI: https://doi.org/10.1007/BF02783478

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