A partially purified chlorophyllase, obtained from alga Phaeodactylum tricornutum, was assayed for its hydrolytic activity towards the pheophytin in ternary micellar systems of hexane/Tris-HCl/surfactant. A wide range of surfactants, sorbitans (Span 20, 40, 60, 80 and 85) and polysorbates (Tween 20, 40, 60, 80 and 85), was used. The use of either 50 $ mu$M of Span 85 or 1 $ mu$M of Tween 80 increased the hydrolytic activity of chlorophyllase by 110 and 23%, respectively. The optimum values of pH, enzyme content, incubation time and temperature for the hydrolytic activity of chlorophyllase were determined as 8.25, 8 $ mu$g protein/ml, 60 min and 27.5$ sp circ$C, respectively. The enzyme was assayed for its hydrolytic activity in the most appropriate ternary system containing Span 85 with purified pheophytin, as well as chlorophyll derivatives, as substrates. Moreover, the values of $V sb{ rm max}/K sb{ rm m}$ ratio for chlorophyllase, using the partially purified pheophytin as substrate, in ternary systems with Span 85 and Tween 80 as surfactants, were 0.15 and 0.08, respectively; however, the value of $V sb{ rm max}/K sb{ rm m}$ ratio for the enzyme, in the ternary system with Span 85, using purified pheophytin as substrate was 0.07. The addition of optimized amounts of individual membrane lipids, L-$ alpha$-phosphatidylcholine, L-$ alpha$-phosphatidyl-DL-glycerol and $ beta$-carotene increased the hydrolytic activity of chlorophyllase, using partially purified pheophytin as substrate, by 50, 36 and 10%, respectively, for Span 85, and 30, 48 and 15%, respectively, for Tween 80; in addition, these lipids increased the enzyme activity by 6, 23 and 31%, respectively, in the Span 85 media, using purified pheophytin as substrate. Phytol showed a competitive inhibitory effect on chlorophyllase activity in both Span and Tween systems containing partially purified pheophytin substrate; however, phytol had an uncompetitive inhibitory effect on the enzyme activity in the S
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.24039 |
| Date | January 1996 |
| Creators | Samaha, Hiba. |
| Contributors | Kermesha, S. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Food Science and Agricultural Chemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001538843, proquestno: MM19849, Theses scanned by UMI/ProQuest. |
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