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From Pseudomonas Fmgi CRDA 037 Characterization of Purified Extracellular Lipase Fractions from Pseudomonas fmgi CRDA 037 by Aliar Abdul Wahrb A thesis submitted to the Faculty of Gnduate Studies and Rtseareh in partial fulîiiment of the rquirements of the degree of Muter of Science O~liaaAbdul Wahab Department of Food Science and Agncultural Chemistry McGill University Montreal, Qu&= National Library Bibliothèque nationale 1+1 ,,nada du Canada Acquisitions and Acquisitions et Bibliographic Services senfices bibliographiques 395 Wellington Street 395, rw Wdlington OttawaON K1AON4 OnawaON K1AW canada canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant a la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la foxme de microfiche/fiim, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in ths thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts kom it Ni la thèse ni des extraits substantiels may be printed or othenvise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. SHORT TITLE EXTRACELLULAR LIPASES FROM PSEUDOMONAS FRQGI ABSTRACT M. Sc. Aliar Abdul Wabab The partially purified extracellular lipase fkom Pseudornoms fiagîCRDA 037, obtained by ammonium sulfate prccipitation, was subjected to iùrther purification by successive ion-exchange and sue-exclusion chromatographies using the Fast Protein Liquid Chromatography system. The purification of the partially purified lipase resulted in two enzymatic fiactions, FIVa' and FIVb', with a purification-fold of 169 and 295, respectively. Native electrophoretic analyses revded the presence of three bands for fiaction 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 fiactions, FNa' and FM', 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 rnM and the V,, values were 2.78 and 2.09 Ulmg, respectively, using triacetin as a substrate. The purified lipase fiactions retained more than 90% of their activity when stored at room temperature for 36 h. The lipase activity of the purified lipase fiactions was compietely inhibited by 10 mM of FeC12, FeC13 and Eliman's ragent. However, 10 mM of CaCIz and EDTA activated the two purified lipase fiactions by 20 to Both fiactions 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 aimost the same extent, the hydrolysis of butter and olive, canola and fish oils. The gas-liquid chromatography analysis of fke 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 chah lengths of C 12 to C 18 whereas fiaction FIVb' showed a non-specific hydrolyzing activity towards fatty acid esters. RÉSUMÉ M. Sc. AIUt Abdul Wahib La lipase partiellement purifiée de Pseudornoltos Fagi CRDA 037, obtenue par précipitation au sulfate d'ammonium, a été davantage purifiée par chromatographies échangeuses d'ions a de tamisage moléculaire a utilisant le système Fast Protein Liquid Chromatography. Deux fhctions purifiées ont été obtenues, FIVa' et FIVb', avec un facteur de purification de 169 a 195, respectivement. Les analyses d'électrophorèse native ont révélé la présence de trois bandes pour k MonFIVéî', avec des poids moléculaires (MW) estimés de 16,2, 25'8 et 38,s kDa et de deux bandes pour FIVb', avec des MW estimés de 15,2 et 25'8 kDa, respectivement. Les deux nanMns purifiées, FIVa' a Fm', ont montré un pH optimum à 9.5 a 10,0, respectivement, et une température de 80°C. Les valeurs de Km pour FNa' et FIVb' ont été de 3,85 et 5'49 mM et les valeurs de V, ont été de 2-78 et 2,09 Ulrng, respectivement, en utilisant la tracétine comme substrat. Après avoir stockée a température ambiante pour une période de 72 h, les fiactions lipasiques purifiées ont conservé plus de 90 ./. de leur activité pendant 36 h. L'activité lipasique des fiactions purifiées a étt5 compléternent inhibée par FeC12, FeCl3 et le réactif dYEllmanà 10 mM. Cependant, CaC12 a EDTA ont activé l'activité des fiactions purifiées de 20 a 50 %. Les deux fractions ont démontré une forte spécificité vis-à-vis des esters de triglycérides a courtes et a longues chaînes. La fiaction FIVa' a montré une forte spécificité envers la tnacétine, la trimyristine et la trioléine. De plus, les deux &actions purifiées ont catalysé l'hydrolyse de beurre et d'huiles d'olive, de canola et de poisson pratiquement au même degré. Les analyses de chromatographie en phase gazeuse d'acides gras libérés par l'hydrolyse des quatre huiles comestibles ont montré que la fraction FIVa' étaiat spécifique pour les esters d'acides gras a longues chaînes de C12:O à Cl8:O alors que la fraction FIVb' etait non-spécifique, hydroplysant aussi bien les esters d'acides gras à longues chaînes saturées et insaturés. I would like to thank my supervisor, Dr. Seh Kennasha, for the guidance, suppofi and patience he offered throughout my study. 1 would iike to extend my gratitude to Dr. André Morin for his numerous advises and his arrangement for the biomass production at the Agriculture Agri-Food Center, CRDA 1 would aiso like to thank Anwer, Marie-Odile and Tami for their assistance. Also special thanks to Pierre-Yves and Wigdan for their valuable advises and Xavier for writing the résumé. And many thanks to al1 my colleagues for their fnendship and good spirit. 1 would like to thank my husband, Osama, my sister, Dalia, my brother, Qussay, my linle sister, Noor, and especiaily my parents for their moral qqmn. TABLE OF CONTENTS LIST OF FIGURES.................................................................................................. ..VIII... 1, INTRODUCTION ..................................................................................................... 1 2- LITERATURE REVIEW .............m~~o~m~ooa~~~o~~o~~~~~~~~~-~moa~m~~~m~ma~oa~a~~a~o--~~~~~~a~~~~ao~~~oa~oam~~o4 2.1. Flavor Production Biotechnology .............................................................................. 4 2.2. Pseudomonas fiagi .................................................................................................. -6 2.2. I . Physical Features ............................................................................................. 6 2.2.2. FhrPra&ct~on by Pseudomomtsfiagrgr ......................................................... 6 2.2.3. Mechism of Flavor Production by P .fiagrgr ..................................................... 7 2.3. Lipases .................................................................................................................... -8 2.3.1. Nomencluture ................................................................................................... 8 2.3.2. Sources of Lzpes ............... .... ................................................................. 9 2.3.3. Characteristics of Lipes-............................................................................. 10 2.3.4 Production of Microbiai Lipares................. .... ............................................. 12 2.3.5. LipeKinetics ................................................................................................ 13 2.3.6. Lipaw Substrores .............. ... ....................................................................... I5 2.3.7. Assay of Lipaw Activity ................................................................................... 16 2.3.7.1. Titrimetric Method .................................................................................... 16 2.3 .7.2. Spectrophotometric Method ...................................................................... 17 2.3.7.3. Fluorimetric Method .................................................................................. 18 2.3.8. LipeSpecr/ciiq ............................................................................................. 18 2.3 .8.1. Positional Specificity ................................................................................. 19 2.3.8.2. Fatty Acid Specificity .................... .. ..................................................... 19 2.3.8.3. Stereoselectivity ....................................................................................... -20 2.3.9. Punijication of li'es..................................................................................... 21 2-3.10. Industrial Application of Lipes................................................................... 23 3. MATERIALS AND METHODS............................................................................. 25 3.1. Bacterial Strain ....................................................................................................... 25 3.2. Chemicals ............................................................................................................... 25 3.3. Biomass
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