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1	Functional characterisation of a novel ferulic acid esterase from Malawian hot spring metagenome Ngobeni, Rhulani January 2011 (has links) <p><!--[if gte mso 9]><xml> <o:OfficeDocumentSettings> <o:AllowPNG /> </o:OfficeDocumentSettings> </xml><![endif]--><!--[if gte mso 9]><xml> <w:WordDocument> <w:View>Normal</w:View> <w:Zoom>0</w:Zoom> <w:TrackMoves /> <w:TrackFormatting /> <w:PunctuationKerning /> <w:ValidateAgainstSchemas /> <w:SaveIfXMLInvalid>false</w:SaveIfXMLInvalid> <w:IgnoreMixedContent>false</w:IgnoreMixedContent> <w:AlwaysShowPlaceholderText>false</w:AlwaysShowPlaceholderText> <w:DoNotPromoteQF /> <w:LidThemeOther>EN-ZA</w:LidThemeOther> <w:LidThemeAsian>X-NONE</w:LidThemeAsian> <w:LidThemeComplexScript>X-NONE</w:LidThemeComplexScript> <w:Compatibility> <w:BreakWrappedTables /> <w:SnapToGridInCell /> <w:WrapTextWithPunct /> <w:UseAsianBreakRules /> <w:DontGrowAutofit /> <w:SplitPgBreakAndParaMark /> <w:EnableOpenTypeKerning /> <w:DontFlipMirrorIndents /> 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,&quot / sans-serif&quot / ">There has been a decline in the global fossil fuel reserves, due to an increasing demand for petroleum. Biofuels can be used as an alternative source of energy whereby biomass is converted to liquid fuels such as bioethanol. There is considerable interest in lipolytic enzymes because of their broad substrate range and for this purpose these enzymes have potential in a variety of biotechnological application. Lipolytic enzymes include esterases (E.C 3.1.1.1) and lipases (E.C3.1.1.3). Esterases preferentially hydrolyse short chain (&lt / C10) ester-containing molecules that are partly soluble in water, while lipases hydrolyse a broad range of substrates preferably water-insoluble fatty acyl molecules (&gt / C10). The aim of this study was to express, purify and characterise the lipolytic enzyme present on afosmid, Try 11, previously isolated from a metagenomic library of a Malawian hotspring, which conferred activity on tributyrin and ethyl ferulate. Bioinformatic analysisof the fosmid insert sequence predicted an open reading frame consisting of 951 bp,designated RHgene34, encoding a 317 amino acid protein with 41 % similarity to the</span><span style="font-size: 10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / mso-fareast-font-family:ArialOOEnc">&alpha / /&beta / hydrolase fold-3 domain protein of </span><i><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">Burkholderia </span></i><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">sp. The RHgene34 protein </span><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">contains conserved motifs of esterases/lipases, such as HGGG (residues 95-98),GxSxG (residues 167 - 171) and the putative catalytic triad composed of Ser157,Asp255 and His285. The gene was cloned and expressed in pET21a(+), and transformed into <i>Escherichia coli </i>Rosetta. <i>p</i>-Nitrophenol (<i>p</i>-Np) fatty acyl esters of different carbon chain lengths were used for kinetic characterisation of RHgene34. </span><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">Kinetic analysis revealed that RHgene34 had a broad range activity on the <i>p</i>-Npesters, from C2 - C14. RHgene34 operates optimally at 45 &ordm / C, pH 9.0 and has a half life of 30 mins at 45 &ordm / C. This study demonstrates that functional screening combined with the sequence analysis is a useful approach for isolating novel enzymes from ametagenome.</span></p> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:12.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / "><br type="_moz" /> </span></p> <br /> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:12.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / "><br type="_moz" /> </span></p> <br />
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,&quot / sans-serif&quot / ">There has been a decline in the global fossil fuel reserves, due to an increasing demand for petroleum. Biofuels can be used as an alternative source of energy whereby biomass is converted to liquid fuels such as bioethanol. There is considerable interest in lipolytic enzymes because of their broad substrate range and for this purpose these enzymes have potential in a variety of biotechnological application. Lipolytic enzymes include esterases (E.C 3.1.1.1) and lipases (E.C3.1.1.3). Esterases preferentially hydrolyse short chain (&lt / C10) ester-containing molecules that are partly soluble in water, while lipases hydrolyse a broad range of substrates preferably water-insoluble fatty acyl molecules (&gt / C10). The aim of this study was to express, purify and characterise the lipolytic enzyme present on afosmid, Try 11, previously isolated from a metagenomic library of a Malawian hotspring, which conferred activity on tributyrin and ethyl ferulate. Bioinformatic analysisof the fosmid insert sequence predicted an open reading frame consisting of 951 bp,designated RHgene34, encoding a 317 amino acid protein with 41 % similarity to the</span><span style="font-size: 10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / mso-fareast-font-family:ArialOOEnc">&alpha / /&beta / hydrolase fold-3 domain protein of </span><i><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">Burkholderia </span></i><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">sp. The RHgene34 protein </span><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">contains conserved motifs of esterases/lipases, such as HGGG (residues 95-98),GxSxG (residues 167 - 171) and the putative catalytic triad composed of Ser157,Asp255 and His285. The gene was cloned and expressed in pET21a(+), and transformed into <i>Escherichia coli </i>Rosetta. <i>p</i>-Nitrophenol (<i>p</i>-Np) fatty acyl esters of different carbon chain lengths were used for kinetic characterisation of RHgene34. </span><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">Kinetic analysis revealed that RHgene34 had a broad range activity on the <i>p</i>-Npesters, from C2 - C14. RHgene34 operates optimally at 45 &ordm / C, pH 9.0 and has a half life of 30 mins at 45 &ordm / C. This study demonstrates that functional screening combined with the sequence analysis is a useful approach for isolating novel enzymes from ametagenome.</span></p> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:12.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / "><br type="_moz" /> </span></p> <br /> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:12.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / "><br type="_moz" /> </span></p> <br />
3	Functional characterisation of a novel ferulic acid esterase from malawian hot spring metagenome Ngobeni, Rhulani January 2011 (has links) >Magister Scientiae - MSc / There has been a decline in the global fossil fuel reserves, due to an increasing demand for petroleum. Biofuels can be used as an alternative source of energy whereby biomass is converted to liquid fuels such as bioethanol. There is considerable interest in lipolytic enzymes because of their broad substrate range and for this purpose these enzymes have potential in a variety of biotechnological application. Lipolytic enzymes include esterases (E.C 3.1.1.1) and lipases (E.C3.1.1.3). Esterases preferentially hydrolyse short chain (<C10) ester-containing molecules that are partly soluble in water, while lipases hydrolyse a broad range of substrates preferably water-insoluble fatty acyl molecules (>C10). The aim of this study was to express, purify and characterise the lipolytic enzyme present on afosmid, Try 11, previously isolated from a metagenomic library of a Malawian hotspring, which conferred activity on tributyrin and ethyl ferulate. Bioinformatic analysisof the fosmid insert sequence predicted an open reading frame consisting of 951 bp,designated RHgene34, encoding a 317 amino acid protein with 41 % similarity to theα/β hydrolase fold-3 domain protein of Burkholderia sp. The RHgene34 protein contains conserved motifs of esterases/lipases, such as HGGG (residues 95-98),GxSxG (residues 167 - 171) and the putative catalytic triad composed of Ser157,Asp255 and His285. The gene was cloned and expressed in pET21a(+), and transformed into Escherichia coli Rosetta. p-Nitrophenol (p-Np) fatty acyl esters of different carbon chain lengths were used for kinetic characterisation of RHgene34. Kinetic analysis revealed that RHgene34 had a broad range activity on the p-Npesters, from C2 - C14. RHgene34 operates optimally at 45 ºC, pH 9.0 and has a half life of 30 mins at 45 ºC. This study demonstrates that functional screening combined with the sequence analysis is a useful approach for isolating novel enzymes from ametagenome. Biofuels Lipolytic enzyme Esterase Lipase Esters
4	Produkce lipolytických enzymů kvasinkami / Production of lipolytic enzymes by yeasts Bradáčová, Kristína January 2018 (has links) This diploma thesis is focused on controlled production of lipolytic enzymes, bioactive substances and lipids by carotenogenic yeasts. Theoretical part deals with characterization of lipolytic enzymes, carotenoids, lipids and their properties, possibility of production and application. In experimental part the enzymes, carotenoids and lipids were produced by red yeasts Rhodotorula mucilaginosa, Cystofilobasidium macerans and Sporidiobolus salmonicolor by submerged cultivation in mineral medium with different additions: glucose, glycerol, fat, fat with glucose, fat with polysorbate 80, fat with glycerol, fat with polyethylene glycol, fat with higher and lower addition of palmitic acid, enzymatic fat hydrolysate, acidic hydrolysate a basic hydrolysate. The activity of extracellular lipase was monitored in medium after 96-hour cultivation. Concentration of -carotene, total carotenoids, ergosterol and ubiquinone was determined by HPLC, concentration of fatty acids and amount of fat by GC. Production had differed depending on used yeasts and substrate. As the best producer of carotenoids Cystofilobasidium macerans was found, ergosterol was highly produced by Sporidiobolus salmonicolor. The production of ubiquinone was almost equivalent in all yeasts and lipolytic activity was the highest in Sporidiobolus salmonicolor. The patricular medium sample with high lipolytic activity was further separated and analysed by ultrafiltration and PAGE-SDS electrophoresis. This diploma thesis was done within the international project ,,LipoFungi“.
5	Etude et validation de nouveaux biomarqueurs pour le diagnostic de la tuberculose pulmonaire / Study and validation of new biomarkers for the diagnosis of pulmonary tuberculosis Akue Brust, Belinda 28 June 2011 (has links) La tuberculose (TB) est une maladie infectieuse causée par M. tuberculosis. En 2009, la mortalité était élevée avec 1,7 millions de décès enregistrés dans le monde. La co-infection par le VIH, notamment en Afrique, et les tuberculoses à bacilles multi-résistants aux antibiotiques, rendent la maîtrise de la pandémie encore plus complexe. Les tests actuels de diagnostic présentent des lacunes notamment en terme de sensibilité pour la microscopie qui est le test le plus utilisé, ou en terme de praticité, en ce qui concerne la culture qui est le test de référence. Le développement de nouveaux tests pour le diagnostic de la TB active représente un enjeu majeur de nos sociétés modernes. Les tests immunologiques, en particulier les tests sérologiques actuellement disponibles, présentent une alternative aux tests bactériologiques couramment utilisés mais leurs performances restent faibles. Pour cela, il est indispensable de cibler de nouveaux biomarqueurs pour développer un test efficace, sensible, rapide et peu onéreux. C'est dans de cadre que s'inscrit ce travail de thèse.Nous rapportons dans ce mémoire, la recherche de marqueurs innovants dans le cadre de la mise au point d'un test de diagnostic de la tuberculose. Nous avons ciblé plusieurs marqueurs protéiques (OmpATb, LipY, Rv0183, Rv1984c et Rv3452) et un marqueur glycolipide, le tréhalose-6,6'-dimycolate (TDM). Parmi ces candidats, plusieurs présentent de réelles performances sur le plan diagnostique. Parallèlement à cette étude, nous avons mis au point une technique de séparation des différentes formes de TDM. La séparation des différentes formes de TDM devrait permettre de déterminer de façon précise les fonctions de ces composants majeurs de la paroi de M. tuberculosis et d'évaluer leur potentiel en terme de diagnostic.Mots clés : tuberculose, sérodiagnostic, détection antigène, détection anticorps, enzymes lipolytiques, glycolipide. / Tuberculosis (TB) is an infectious disease caused by M. tuberculosis. In 2009, mortality was high with 1,7 million of recorded deaths in the world. The co-infection by the HIV, in particular in Africa, and tuberculosis with multi-resistant bacilli to antibiotics, make the control of pandemia more complex. The current tests of diagnosis present gaps, in particular, in term of sensitivity for microscopy, which is currently the used test, or in term of praticity, for the culture which is considered as the gold standard.The development of new tests for the diagnosis of TB active represents a major challenge of our modern societies. The immunological tests, in particular the serologic tests available, can present an alternative to the currently bacteriological tests. However, their performances are not sufficient. That's why, it is essential to target new biomarkers which permit to establish an effective, sensitive, fast and cheap test. It is in this context that my study takes place.We report in this memory, the research of innovating markers for the development of a diagnostic test of tuberculosis. We targeted several protenic markers (OmpATb, LipY, Rv0183, Rv1984c and for Rv3452) and a glycolipid marker, the trehalose-6,6'-dimycolate (TDM). Among these candidates, several shown good performances for active TB diagnosis. In parallel to this study, a technique of separation of the various forms of TDM was developed. The separation of the various forms of TDM will permit to study their role on the biological activities and evaluate their potential in term of diagnosis.Key words: tuberculosis, serodiagnostic, antigen detection, antibody detection, lipolytic enzymes, glycolipid. Tuberculose Sérodiagnostic Détection antigène Détection anticorps Enzymes lipolytiques Glycolipide Tuberculosis Serodiagnosis Antigen detection Antibody detection Lipolytic enzyme Glycolipid

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