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dc.contributor.authorSalameh, Moh'd A.
dc.date.accessioned2014-03-04T01:11:49Z
dc.date.available2014-03-04T01:11:49Z
dc.date.issued2006-08
dc.identifier.othersalameh_mohd_a_200608_phd
dc.identifier.urihttp://purl.galileo.usg.edu/uga_etd/salameh_mohd_a_200608_phd
dc.identifier.urihttp://hdl.handle.net/10724/23507
dc.description.abstractThermosyntropha lipolytica DSM 11003 is an anaerobic thermophilic alkalitolerant bacterium which grows syntrophically with a methanogen on lipids utilizing only the fatty acid moieties, but not the glycerol. Since no lipases from thermophilic anaerobes have been analyzed, there is the possibility to find enzymes with different stereo and substrate specificity not observed among the Gram negative aerobic ones. The objective of this study was to characterize the lipases that are produced by this bacterium with the intent of producing commercially viable enzymes for use in high temperature, high pH-value applications, and various organic synthesis reactions such as structured lipids, remodeling acyl-alcohols and the resolution of racemates. Two enzymes, termed LipA and LipB, were purified from the culture supernatant to gel electrophoretic purity by ammonium sulfate precipitation and column chromatography using Octyl Sepharose fast flow. The apparent molecular weight of LipA and LipB determined by SDS-PAGE were 50 and 57 kDa, respectively. The temperature optima of purified LipA and LipB was 96 °C, this is the highest among all known lipases so far. The pHoptima of LipA and LipB were 9.4 and 9.6, respectively. They are among the most thermostable lipases with a half life of 24 h at 75 °C. Both enzymes are true lipases that are interfacially activated by the presence of insoluble substrates. They prefer glycerides with long chain fatty acids (C12 to C18), and prefer to hydrolyse ester bonds at the primary position. In addition, the effect of different detergents on enzymes activity and stability was investigated. SDS was found to have the highest impact on both lipases by enhancing the activity of both LipA and LipB by approximately 9 folds. Assay analyses using the serine inhibitor E600 with increasing concentration of SDS and Tween 20 strongly suggest that SDS and Tween 20 promote conformational changes by binding to the lid domain and/or active site pocket so that the site becomes accessible to the substrate. The purified native lipases showed a strong tendency to form catalytically active oligomers as observed by gel filtration chromatography. This property might be a major contributor to their high thermostability. Finally, the ability of both lipases to conduct reverse synthesis reactions was investigated. The maximum catalytic activity was measured at 85°C in isooctane. Octyl oleate and lauryl oleate were the highest conversion products of the esterefication reactions. In addition, LipA and LipB effectively catalyzed the synthesis of diacylglycerols, in particular 1,3-dioleoyl glycerol.
dc.languageeng
dc.publisheruga
dc.rightspublic
dc.subjectThermosyntropha lipolytica
dc.subjectAlkalithermophilic
dc.subjectLipases
dc.subjectgel filtration
dc.subjectprotein oligomerization
dc.subjectlipase purification
dc.subjectthermostability
dc.subjectthermal activity
dc.subjectp-nitrophenyl laurate
dc.subjectSDS
dc.subjectcritical micelle concentration
dc.subjectE600
dc.subjectTween 20
dc.subjectdetergents
dc.subjectregiospecifi
dc.titleAlkalithermophilic lipases from Thermosyntropha lipolytica
dc.typeDissertation
dc.description.degreePhD
dc.description.departmentMicrobiology
dc.description.majorMicrobiology
dc.description.advisorJuergen Wiegel
dc.description.committeeJuergen Wiegel
dc.description.committeeWilliam B. Whitman
dc.description.committeeRobert Maier
dc.description.committeeAnna Karls
dc.description.committeeMichael W. Adams


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