Biomass production of chlorophyllase of the marine alga (Phaeodactylum tricornutum) at the exponential and stationary stages was performed. The results demonstrated that the biomass yield at the stationary stage was three times that of the exponential stage. A procedure for the extraction and purification of chlorophylls from fresh spinach leaves, used as substrate, was also developed. Chlorophyllase was extracted from photosynthetic membranes of the disrupted cells and partially purified. The purification procedure resulted into 70-fold increase in enzyme activity. Further purification of the partially purified enzyme was performed, using preparative isoelectric focusing on Rotofor-Cell System. Three enzyme fractions, FI$ sp prime$, FII$ sp prime$, and FIII$ sp prime$ were separated, however, most of enzyme activity (84%) was located in fraction FII$ sp prime$. The partially purified chlorophyllase was further purified by native preparative gel electrophoresis on Prep-Cell System, which resulted into a single active fraction. The purified enzyme fraction was then subjected to further purification, using automated Fast Protein Liquid Chromatography (FPLC) System, on ion-exchange Mono Q HR 5/5 column. The purification procedure resulted into two well separated isozymes, FI$ sp prime$ and FII$ sp prime$. Enzyme fraction (FI$ sp prime$) showed the highest enzymatic activity compared to FII$ sp prime$. The homogeneity of each fraction was demonstrated by a single protein band on SDS-PAGE. The molecular weights of these fractions FI$ sp prime$ and FII$ sp prime$ were 67 kD and 66kD, respectively. The optimum pH for chlorophyllase activity fractions FI$ sp prime$, and FII$ sp prime$ were 8.0 and 8.3, respectively. The enzymatic fraction FI$ sp prime$ showed higher activity towards commercial purified chlorophyll b when it was compared to that with the crude chlorophyll, partially purified chlorophyll and commercial purified chlorophyll a. However, enzymatic fraction FI
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.41110 |
| Date | January 1993 |
| Creators | Khalyfa, Abdelnaby |
| Contributors | Kermasha, 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 | Doctor of Philosophy (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: 001338493, proquestno: NN87859, Theses scanned by UMI/ProQuest. |
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