The partially purified extracellular lipase from Pseudomonas fragi CRDA 037, obtained by ammonium sulfate precipitation, was subjected to further purification by successive ion-exchange and size-exclusion chromatographies using the Fast Protein Liquid Chromatography system. The purification of the partially purified lipase resulted in two enzymatic fractions, FIVa ' and FIVb', with a purification-fold of 169 and 195, respectively. Native electrophoretic analyses revealed the presence of three bands for fraction FIVa', with estimated molecular weights (MW) of 16.2, 25.8 and 38.5 kDa and two bands for FIVb' , with estimated MW of 15.2 and 25.8 kDa. The two purified fractions, FIVa' and FIVb', showed an optimum pH of 9.5 and 10.0, respectively, and an optimum temperature of 80°C. The Km values for FIVa' and FIVb' were 3.85 and 5.49 mM and the V max values were 2.78 and 2.09 U/mg, respectively, using triacetin as a substrate. The purified lipase fractions retained more than 90% of their activity when stored at room temperature for 36 h. The lipase activity of the purified lipase fractions was completely inhibited by 10 mM of FeCl 2, FeCl3 and Ellman's reagent. However, 10 mM of CaCl2 and EDTA activated the two purified lipase fractions by 20 to 50%. Both fractions exhibited high specificity towards short- and long-chain fatty acid esters of triacylglycerols. Fraction FIVa' showed higher specificity towards triacetin, tristearin and tripalmitin, whereas fraction FIVb' exhibited higher activity with triacetin, trimyristin and triolein. In addition, the two purified lipase fractions were able to catalyze, to almost the same extent, the hydrolysis of butter and olive, canola and fish oils. The gas-liquid chromatography analysis of free fatty acids, obtained by the hydrolysis of the four edible oils, revealed that fraction FIVa' was more specific for the hydrolysis of fatty acid esters chain lengths of C12 to C18 whereas fraction FIVb' showed a non-specific hydrolyzin
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.21498 |
| Date | January 1999 |
| Creators | Abdul Wahab, Aliaa. |
| 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 | 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: 001659857, proquestno: MQ50703, Theses scanned by UMI/ProQuest. |
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