Pseudomonas fragi CRDA 037 was used to produce both intracellular and extracellular lipases. The crude lipase fractions were fractionated using ammonium sulfate to obtain partially purified intracellular and extracellular lipases; the fraction precipitated at 20-60% of saturation of the intracellular proteins was retained as the source of the endolipase, whereas the culture medium precipitated at 20-40% of saturation, was used as the source of exolipase. Native gel electrophoresis (PAGE) suggested that the partially purified extracellular lipase has a molecular weight of 25,500 Da. Three major electrophoretic bands were present in the intracellular lipase fraction at 70,000, 49,000, and 35,500 Da. The partially purified lipases were characterized with respect to their optimum pH and temperature for lipase activity, and well as for their kinetics, specificities, and reactions toward inhibitors. The optimum pH for the activity of the endolipase was found to be 9.0 whereas that of the exolipase was 8.75. With respect to optimum temperature, 30$ sp circ$C was determined to be the best for the endolipase while 35$ sp circ$C was the optimal value for the exolipase. Enzyme specificity was carried out using triacetin, tributyrin, trimyristin, and triolein as substrates. The results for the exolipase indicated that the lowest K$ sb{m}$ was obtained with trimyristin and the highest K$ sb{m}$ was obtained with triolein whereas for the endolipase, the highest K$ sb{m}$ was obtained with tributyrin and the lowest was with trimyristin. Experiments carried out with inhibitors indicated that both lipases are serine lipases, sulfydryl enzymes, and that tryptophan is essential for maintaining the conformation of the proteins. The most potent inhibitor was ferrous chloride, and sodium deoxycholate was a weak inhibitor for both lipases, however, it was an activator at low concentrations for the exolipase.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.23425 |
| Date | January 1995 |
| Creators | Schuepp, Catherine |
| Contributors | Kermasha, Selim (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: 001487644, proquestno: MM12267, Theses scanned by UMI/ProQuest. |
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