Succinat und ihre Derivate werden in vielfältiger Weise in den Bereichen Tenside, Lebensmittel, Pharmazeutika und Polymere angewendet. Aufgrund der derzeit kostenintensiven petrochemischen Synthese ist die aerobe nicht-konventionelle Hefe Yarrowia (Y.) lipolytica für die biotechnologische Succinatsynthese von großem Interesse. In der vorliegenden Arbeit wurde das Potential dieser Hefe für eine industrielle Succinatproduktion unter Betrachtung des Einflusses der enzymatischen Aktivitäten von Succinyl-CoA Synthetase und Pyruvat-Carboxylase auf die Succinatsynthese untersucht. Es wurde eine Steigerung der Succinatausbeute um 40 % durch die Erhöhung der Pyruvat-Carboxylase Aktivität um den Faktor 7-8 gemeinsam mit der Deletion des Gens der β Untereinheit der Succinyl-CoA Synthetase im genetisch veränderten Y. lipolytica Stamm H222-AK10 (mcPYC Δscs2) erzielt. Unter Verwendung von Glycerol als C-Quelle wurde eine Erhöhung der Succinatbildung der Transformande H222 AK10 im Vergleich zum Wildtyp von 5,1 ± 0,7 g/l auf 8,7 ± 1,6 g/l nachgewiesen. Die Raum-Zeit-Ausbeute dieses Hefestammes verdoppelte sich von 11,9 ± 1,3 mg/l*h auf 21,9 ± 2,5 mg/l*h. Eine Erhöhung der Sekretion organischer Säuren gelang hingegen nicht durch den alleinigen Verlust der Succinyl-CoA Synthetase Aktivität in den Stämmen H222-AK4 (scs1::URA3), H222-AK8 (scs2:.URA3) und H222-AK9 (scs1::URA3 Δscs2) oder durch die alleinige Aktivitätserhöhung der Pyruvat-Carboxylase in H222-AK1 (mcPYC). Des Weiteren wurde ein Y. lipolytica Stamm erzeugt, der durch die Überexpression der für die Succinyl-CoA Synthetase kodierenden Gene SCS1 und SCS2 charakterisiert ist. Die Transformande H222 AK2 (mcSCS1 mcSCS2) bildete unter den gleichen Kultivierungsbedingungen durchschnittlich 2 g/l weniger Succinat als der Wildtyp (5,1 ± 0,7 g/l). Auch die zusätzliche Erhöhung der Pyruvat-Carboxylase Aktivität um den Faktor 4 in der Transformande H222 AK3 (mcPYC mcSCS1 mcSCS2) konnte den negativen Effekt der erhöhten Gen-Dosen von SCS1 und SCS2 auf die Succinatsynthese nicht aufheben. Dementsprechend wurden für H222-AK3 eine Succinatausbeute von 3,1 ± 0,3 g/l bestimmt.

Succinat und ihre Derivate werden in vielfältiger Weise in den Bereichen Tenside, Lebensmittel, Pharmazeutika und Polymere angewendet. Aufgrund der derzeit kostenintensiven petrochemischen Synthese ist die aerobe nicht-konventionelle Hefe Yarrowia (Y.) lipolytica für die biotechnologische Succinatsynthese von großem Interesse. In der vorliegenden Arbeit wurde das Potential dieser Hefe für eine industrielle Succinatproduktion unter Betrachtung des Einflusses der enzymatischen Aktivitäten von Succinyl-CoA Synthetase und Pyruvat-Carboxylase auf die Succinatsynthese untersucht. Es wurde eine Steigerung der Succinatausbeute um 40 % durch die Erhöhung der Pyruvat-Carboxylase Aktivität um den Faktor 7-8 gemeinsam mit der Deletion des Gens der β Untereinheit der Succinyl-CoA Synthetase im genetisch veränderten Y. lipolytica Stamm H222-AK10 (mcPYC Δscs2) erzielt. Unter Verwendung von Glycerol als C-Quelle wurde eine Erhöhung der Succinatbildung der Transformande H222 AK10 im Vergleich zum Wildtyp von 5,1 ± 0,7 g/l auf 8,7 ± 1,6 g/l nachgewiesen. Die Raum-Zeit-Ausbeute dieses Hefestammes verdoppelte sich von 11,9 ± 1,3 mg/l*h auf 21,9 ± 2,5 mg/l*h. Eine Erhöhung der Sekretion organischer Säuren gelang hingegen nicht durch den alleinigen Verlust der Succinyl-CoA Synthetase Aktivität in den Stämmen H222-AK4 (scs1::URA3), H222-AK8 (scs2:.URA3) und H222-AK9 (scs1::URA3 Δscs2) oder durch die alleinige Aktivitätserhöhung der Pyruvat-Carboxylase in H222-AK1 (mcPYC). Des Weiteren wurde ein Y. lipolytica Stamm erzeugt, der durch die Überexpression der für die Succinyl-CoA Synthetase kodierenden Gene SCS1 und SCS2 charakterisiert ist. Die Transformande H222 AK2 (mcSCS1 mcSCS2) bildete unter den gleichen Kultivierungsbedingungen durchschnittlich 2 g/l weniger Succinat als der Wildtyp (5,1 ± 0,7 g/l). Auch die zusätzliche Erhöhung der Pyruvat-Carboxylase Aktivität um den Faktor 4 in der Transformande H222 AK3 (mcPYC mcSCS1 mcSCS2) konnte den negativen Effekt der erhöhten Gen-Dosen von SCS1 und SCS2 auf die Succinatsynthese nicht aufheben. Dementsprechend wurden für H222-AK3 eine Succinatausbeute von 3,1 ± 0,3 g/l bestimmt.:Inhaltsverzeichnis Abbildungsverzeichnis VI Tabellenverzeichnis IX Abkürzungsverzeichnis XI 1. Einleitung 1 1.1. Bernsteinsäure, Succinat 1 1.2. Succinat als Zwischenprodukt des Tricarbonsäurezyklus 3 1.2.1. Die Enzyme des TCC in Saccharomyces cerevisiae 5 1.2.2. Succinatbildung im TCC durch die Succinyl-CoA Synthetase 6 1.2.3. Pyruvat-Carboxylase 7 1.3. Succinat als Endprodukt des Glyoxylatzyklus 9 1.4. Biotechnologische Herstellung von Succinat 10 1.4.1. Succinatproduktion mit Actinobacillus succinogenes und Anaerobiospirillum succiniproducens 11 1.4.2. Succinatproduktion mit Corynebacterium glutamicum 12 1.4.3. Succinatproduktion mit Escherichia coli 12 1.4.4. Yarrowia lipolytica als potentieller Succinatproduzent 15 1.5. Yarrowia lipolytica 15 1.6. Zielstellung 18 2. Material und Methoden 20 2.1. Geräte 20 2.2. Chemikalien, Biochemikalien und Nukleinsäuren 21 2.2.1. Feinchemikalien 21 2.2.2. Enzyme 22 2.2.3. Verbrauchsmaterialien und Kitsysteme 23 2.3. Verwendete Plasmide 23 2.4. Konstruierte Plasmide 24 2.5. Oligonukleotide 25 2.6. Mikroorganismen 26 2.6.1. Escherichia coli 26 2.6.2. Yarrowia lipolytica 27 2.7. Kultivierung 27 2.7.1. Kultivierung von Escherichia coli 27 2.7.2. Kultivierung von Yarrowia lipolytica 28 2.8. Gentechnische Methoden 29 2.8.1. Genomische DNA-Isolierung 29 2.8.2. Agarose-Gelelektrophorese 29 2.8.3. Polymerase Kettenreaktion 30 2.8.4. Verdau der DNA mit Restriktionsendonukleasen 31 2.8.5. DNA-Aufreinigung 31 2.8.6. Plasmidisolierung aus Escherichia coli 31 2.8.7. Dephosphorylierung 31 2.8.8. Ligation 32 2.8.9. Transformation elektrokompetenter Escherichia coli Zellen 32 2.9. Plasmidkonstruktion 33 2.9.1. Konstruktion der Expressionskassette für die Überexpression des Pyruvat-Carboxylase kodierenden Gens 33 2.9.2. Konstruktion der Expressionskassetten für die Überexpression der Gene der α- und β-Untereinheit der Succinyl-CoA Synthetase 34 2.9.3. Konstruktion der Deletionskassette des für die α-Untereinheit der Succinyl-CoA Synthetase kodierenden Gens 35 2.9.4. Konstruktion der Deletionskassette des für die β-Untereinheit der Succinyl CoA Synthetase kodierenden Gens 36 2.9.5. Sequenzierung konstruierter Plasmide 36 2.10. Transformation von Yarrowia lipolytica Zellen 37 2.10.1. Transformation von Yarrowia lipolytica mittels der LiAc-Methode 37 2.10.1.1. Herstellung chemisch kompetenter Yarrowia lipolytica Zellen 37 2.10.1.2. Transformation chemisch kompetenter Hefezellen 37 2.10.2. Herstellung elektrokompetenter Yarrowia lipolytica Zellen 38 2.10.3. Elektrotransformation von Yarrowia lipolytica 38 2.11. Southern Blot 39 2.11.1. Transfer der DNA auf eine Nylonmembran 39 2.11.2. Sondenherstellung 39 2.11.3. Immunologische Detektion 40 2.11.4. Strippen der Southern Blot Membran 40 2.12. Biochemische Methoden 41 2.12.1. Ernte und Aufschluss der Hefezellen 41 2.12.2. Aktivitätsbestimmung der Citrat-Synthase 41 2.12.3. Aktivitätsbestimmung der Aconitase 41 2.12.4. Aktivitätsbestimmung der NADP-abhängige Isocitrat-Dehydrogenase 42 2.12.5. Aktivitätsbestimmung der NAD-abhängige Isocitrat-Dehydrogenase 42 2.12.6. Aktivitätsbestimmung der α-Ketoglutarat-Dehydrogenase 43 2.12.7. Aktivitätsbestimmung der Succinyl-CoA Synthetase 43 2.12.8. Enzymatische Mitochondrienpräparation 46 2.12.9. Aktivitätsbestimmung der Succinat-Dehydrogenase 47 2.12.10. Aktivitätsbestimmung der Fumarase 47 2.12.11. Aktivitätsbestimmung der Malat-Dehydrogenase 48 2.12.12. Aktivitätsbestimmung der Isocitrat-Lyase 48 2.12.13. Aktivitätsbestimmung der Pyruvat-Carboxylase 48 2.12.14. Proteinbestimmung 49 2.13. Mikroskopische Bestimmung der Zellmorphologie 49 2.14. Kultivierung 49 2.14.1. Bestimmung der optischen Dichte 50 2.14.2. Animpfen der Hauptkultur 50 2.14.3. Succinatproduktionsmedium 50 2.14.4. Kultivierung unter Thiaminlimitation 51 2.14.5. Kultivierung unter Stickstofflimitation 52 2.14.6. Bestimmung organischer Säuren mittels Ionenchromatographie 52 2.15. Bioinformatik 53 3. Ergebnisse 54 3.1. Überexpression des Pyruvat-Carboxylase kodierenden Gens 54 3.2. Überexpression der Succinyl-CoA Synthetase kodierenden Gene 58 3.2.1. Bestimmung der Succinyl-CoA Synthetase Enzymaktivität 61 3.2.2. Bestimmung der spezifischen Aktivität weiterer Enzyme 61 3.3. Überexpression der für die Pyruvat-Carboxylase und Succinyl-CoA Synthetase kodierenden Gene 63 3.3.1. Bestimmung der spezifischen Aktivitäten der Enzyme des TCC und der PYC sowie der ICL 66 3.4. Deletion der Succinyl-CoA Synthetase Gene 68 3.4.1. Bestimmung der Succinyl-CoA Synthetase Enzymaktivität 72 3.4.2. Bestimmung weiterer spezifischer Enzymaktivitäten 73 3.5. Überexpression des Gens der Pyruvat-Carboxylase gemeinsam mit der Deletion des Gens für die β Untereinheit der Succinyl-CoA Synthetase 74 3.5.1. Bestimmung der spezifischen Aktivitäten von Enzymen des TCC, sowie der PYC und ICL 76 3.6. Morphologische Veränderungen der Transformanden 78 3.7. Produktion organischer Säuren im Succinatproduktionsmedium 80 3.9.1 Bestimmung der PYC-, ICL- und SDH-Aktivitäten während der Kultivierung im Succinatproduktionsmedium 87 3.8. α-Ketoglutarat-, Pyruvat- und Fumaratproduktion unter Thiaminlimitation 90 3.9. Citrat- und Isocitratproduktion unter Stickstofflimitation 96 4. Diskussion 100 4.1. Auswahl einer geeigneten C-Quelle für die Succinatproduktion 101 4.2. Reduktion der Succinatproduktion von Yarrowia lipolytica 102 4.3. Genetische Veränderungen, für die kein Einfluss auf die Succinatproduktion von Yarrowia lipolytica nachgewiesen wurde 106 4.3.1. Überexpression des Pyruvat-Carboxylase kodierenden Gens 107 4.3.2. Deletion der Succinyl-CoA Synthetase Gene 108 4.4. Erhöhung der Succinatproduktion von Yarrowia lipolytica 112 4.5. Auswirkungen auf die Produktion anderer organischer Säuren 119 4.5.1. α-Ketoglutarat-, Pyruvat- und Fumaratproduktion unter Thiaminlimitation 119 4.5.2. Citrat- und Isocitratproduktion unter Stickstofflimitation 121 4.6. Morphologische Veränderungen 122 4.6.1. Koloniemorphologie 122 4.6.2. Zellmorphologie 124 Literaturverzeichnis 126

Protein phosphorylation constitutes an important mechanism for cellular regulation in both Eucarya and Bacteria. All living organisms evolved from a common progenitor; this implies that protein phosphorylation as a means of regulation also exists in Archaea. Previously, in the sulfur-dependent archaeon Sulfolobus solfataricus a gene was cloned encoding a protein-serine/threonine phosphatase that was similar to eucaryal protein-serine/threonine phosphatases type 1, 2A, and 2B. To identify protein phosphatases in other archaeons, oligonucleotides encoding conserved regions of eucaryal protein-serine/threonine phosphatases were used in the polymerase chain reaction to amplify genomic DNA from the methanogenic archaeon Methanosarcina thermophila. From the PCR reaction a fragment of DNA was isolated that encoded a portion of a protein phosphatase. Using this DNA fragment as a probe, the entire phosphatase gene was isolated. The amino acid sequence of the phosphatase encoded by this gene displayed greater than 30% identity with eucaryal protein-serine/threonine phosphatase type 1. The gene encoding the Methanosarcina phosphatase was expressed in Escherichia coli. The expressed protein exhibited protein serine phosphatase activity that was sensitive to inhibitors of eucaryal phosphatases such as okadaic acid, microcystin, calyculin, and tautomycin. In order to identify potential endogenous substrates of archaeal protein-serine/threonine phosphatases and kinases, a study was initiated to characterize the most prominent phosphoproteins in S. solfataricus. Cell extracts were incubated with [γ-³²P] ATP, MgCl₂, and MnCl₂, and the proteins in the extracts were separated by SDS-PAGE. Autoradiography of the gels revealed four prominent phosphoproteins with apparent molecular masses of 35, 46, and 50 kDa. N-terminal sequence analysis and enzymatic assays of the 35 kDa phosphoprotein identified this phosphoprotein as the a-subunit of succinyl-CoA synthetase. N-terminal sequence analysis and enzymatic assays revealed that the 50 kDa phosphoprotein was a hexosephosphate mutase. Neither the 50 kDa nor the 35 kDa phosphoprotein appeared to be the target of protein kinases or phosphatases. Therefore, while protein-serine phosphatases exist in Archaea, the targets of these phosphatases have yet to be determined. / Ph. D.

Indian Institute Of Science / Proteins associated with metabolic pathways have received considerable attention in an effort to understand the molecular details of the complex reactions catalyzed in vivo. Enzymes belonging to these pathways have also been explored as potential targets for therapeutic intervention. The L-Lysine biosynthesis pathway in particular remains an attractive target for the design of new anti-microbial compounds. The rationale for this interest stems from the finding that the pathway for Lysine biosynthesis is only present in bacteria and is absent in humans. m-DAP/L-Lysine is an essential component of the bacterial cell wall. The L-Lysine biosynthesis pathway in bacteria is fairly diverse. Three major routes for the biosynthesis of m-DAP/L-Lysine are currently known. These are the succinylase, the acetylase and the dehydrogenase pathways. The results reported in this thesis are based on enzymes involved in the L-Lysine biosynthesis pathway of a nosocomial pathogen, Staphylococcus aureus spp. COL. The structural and biochemical characterization of three enzymes, Dihydrodipicolinate synthase (DapA), Dihydrodipicolinate reductase (DapB) and Succinyl diaminopimelate desuccinylase (DapE) provide a mechanistic rationale for the activity of these proteins. These studies reveal substantial differences in the regulatory mechanisms of these enzymes that could potentially be utilized for the design of inhibitors that specifically target this biosynthesis route. This thesis is organized as follows: Chapter 1 provides an introduction to the topic of this thesis. The first part of this chapter describes the characteristic features of Staphylococcus aureus and aspects relating to the identification, pathogenesis and genomic differences in S. aureus strains. The general features of the peptidoglycan layer of the bacterial cell wall including its structure and composition are also discussed. The second part of this chapter provides an overview of the Lysine biosynthesis pathway and details of the enzymes in this pathway. Chapter 2 describes the structural and functional features of Dihydrodipicolinate synthase (DHDPS) also referred to as DapA. DHDPS catalyzes the first committed step of L-Lysine biosynthesis. Here we report the crystal structure of the native and pyruvate complexes of S. aureus DHDPS. S. aureus DHDPS is a dimer, both in solution as well as in the crystal. The functional characterization of S. aureus DHDPS revealed that this enzyme is active as a dimer. This feature distinguishes the S. aureus enzyme from the E. coli homologue where a tetrameric quaternary arrangement is essential for the activity of this protein. A comparison between the native and pyruvate-bound structures also provides a structural basis for the ping-pong reaction mechanism of this enzyme whereby the catalytic triad is drawn closer to facilitate proton transfer upon pyruvate binding. It was also noted that unlike the E. coli homologue, S. aureus DHDPS is not feedback inhibited by lysine. The lack of feedback inhibition in S. aureus DHDPS could be attributed to a unique allosteric site. The different quaternary arrangement and a distinct allosteric pocket in this enzyme thus provide a structural template for the design of specific inhibitors for this enzyme. Chapter 3 is based on preliminary studies of Dihydrodipicolinate reductase (DHDPR), encoded by the dapB gene. DHDPR catalyzes the second committed step in m-DAP/L-Lysine biosynthesis. The dapB gene encoding DHDPR was cloned and over-expressed in E. coli. Two variations of the recombinant protein were examined- one with a hexa-histidine tag at the C-terminus and the other without any tag. The recombinant DHDPR with the C-terminal hexa-histidine tag was purified by Ni-affinity chromatography and was subsequently crystallized. However, data sets collected on these crystals could not be examined further due to pronounced pseudo-translational symmetry and poor resolution. The recombinant DHDPR protein without an expression tag was purified by anion-exchange and size exclusion chromatography. Analytical gel filtration studies with recombinant DHDPR is consistent with a tetrameric quaternary arrangement of DHDPR subunits with a calculated molecular mass of 135 kDa. Diffraction data were collected to 3.3 Å resolution on crystals of apo DHDPR. These crystals belong to the C-centered monoclinic space group (C2) with unit cell parameters a = 63.17 Å, b = 78.91 Å, c = 128.38 Å and γ = 110.0°. Assuming two molecules in the asymmetric unit, the calculated Matthew’s coefficient (Vm) was 2.32 Å3 Da-1 and solvent content was 47.0 %. Molecular replacement (MR) trials with a model combining E .coli DHDPR structures (residues 1-106 of 1ARZ and 108-241 of 1DIH) as a search model resulted in a successful MR solution with two molecules in the asymmetric unit. Chapter 4 is based on the structure and regulatory mechanism of DapE also referred to as Sapep. This enzyme belongs to the M20 family of proteases and is characterized by diverse substrate specificity and multiple functional roles. These include Succinyl diaminopimelate desuccinylase, a Mn2+-dependent di-peptidase and a -lactamase. The chemical reaction involved in all these functions is broadly similar and involves amide bond hydrolysis. In an effort to understand the structure and regulatory features of this enzyme, the structure of Sapep was determined both in a Mn2+-bound form and in a metal-free (apo) form. A comparison between these structures revealed that large inter-domain movements potentially regulate the activity of this enzyme. These structures also revealed an additional regulatory mechanism wherein the inactive conformation is stabilized by a disulfide bond in the vicinity of the active site. Although these cysteines, Cys155 and Cys178 are not active site residues, the reduced form of this enzyme is substantially more active as a peptidase. The characterization of disulfide bond in the apo-form of the protein in solution by mass spectrometric studies and the requirement of a reducing agent for optimal catalytic activity of this protein suggests that the conformational features noted in crystal structures are also likely in solution. The structural and biochemical features of this enzyme thus provide a basis to rationalize the multiple functional roles of this protein with potential applications to MRSA-specific therapeutic strategies. Chapter 5 provides a summary of the biochemical and structural data on the three enzymes of the L-Lysine biosynthesis pathway in S. aureus spp. COL. The emphasis of the discussion in this chapter is on features that are specific to these S. aureus enzymes. The latter part of this chapter is based on the scope of future studies in this area. The appendix sections of this thesis are based on a project involving the catalytic domain of a receptor protein tyrosine phosphatase CRYP-2/cPTPRO. Appendix-I includes the structure-function analysis of the catalytic domain of CRYP-2/cPTPRO. CRYP-2/cPTPRO is a receptor protein tyrosine phosphatase (PTP) that is selectively expressed in neurons and has been implicated in axon growth and guidance. The extracellular receptor domain of this protein has eight fibronectin typeIII repeat regions while the intracellular region consists of a catalytic PTP domain. The crystal structure of CRYP-2 revealed two molecules of the catalytic domain in the asymmetric unit. The substantial buried surface area of this crystallographic oligomer suggested a homo-dimer of the catalytic domain. Solution studies however suggested that this protein is a monomer in solution based on the elution profile of CRYP-2 in a size exclusion chromatography experiment. The monomeric nature of CRYP-2 thus suggests that dimerization induced modulation of enzyme activity, such as that seen in RPTP- where a helix-turn-helix segment of one monomer blocks the active site of the other, is not possible in the case of CRYP-2. Both monomers of CRYP-2 reveal a nitrate ion bound at the active site. An advantage provided by the crystallographic dimer of CRYP-2 was that it allowed us to visualize this protein with its active site lid (WPD-loop) in both the open and closed conformations. A structural comparison of CRYP-2 with other PTP’s suggests that minor conformational rearrangements, as opposed to dimerization, could serve to regulate the activity of members of the type III family of RPTPs. Appendix-II describes a project to examine the feasibility of utilizing mesoporous matrices of alumina and silica for the controlled inhibition of enzymatic activity. In these studies, we employed bare and functionalized mesoporous alumina and MCM48 silica to deliver para-nitrocatechol sulfate (pNCS), a potent competitive PTP inhibitor. pNCS was chosen as model inhibitor because of the ease of monitoring its release using UV-Visible absorption spectroscopy. CRYP-2 was used as model enzyme in this analysis. Inhibition of catalytic activity was examined using the sustained delivery of para-nitrocatechol sulfate (pNCS) from bare and amine functionalized MCM48 and Al2O3. Among the various mesoporous matrices employed, amine functionalized MCM48 exhibited the best controlled release of pNCS and inhibition of CRYP-2. Appendix-3 incorporates additional methodologies and technical details that could not be included in the main body of the thesis.

Growing concerns of the negative effects on the environment and dependency of fossil fuelsare major driving forces for finding novel sustainable production pathways for plastic.Metabolic engineering has emerged as a powerful tool to enable microorganisms to producenon-native metabolites. The aim of this project was recombinant production of 4-hydroxybutyrate (4-HB) by expressing two enzymes in the model organism Escherichia coli.α-ketoglutarate decarboxylase (SucA) from Mycobacterium smegmatis followed by 4-hydroxybutyrate dehydrogenase (4-HBd) from Clostridium kluyveri was expressed inEscherichia coli. Results showed that the genes were successfully transformed and expressedin E. coli and after protein purification a concentration of 0.9 g/L SucA and 9.8 g/L 4-HBdwas achieved. Furthermore, some protein activity was detected by a coupled reaction withSucA and 4-HBd. When the enzymes got coupled together a change in NADH concentrationcould be detected spectrophotometrically. The enzymes were also tested for substratespecificity by using substrates with various carbon chain lengths and a decrease in NADHconcentration was seen. However, a decrease in the negative control for the experiments wasalso seen indicating a breakdown of NADH over time rather than consumption. Therefore, noconclusion could be drawn about the promiscuity of the enzymes. Lastly a single plasmidssystem was tested where both the genes were ligated on the same plasmid (pCDF duet) andexpressed successfully in E. coli Bl21DE3. / Ökad oro för miljön samt behovet av fossila resurser för produktion av plaster har gjort detnödvändigt att skapa nya och mer hållbara produktions vägar. Genetisk modifikation av olikaorganismer har utvecklats som ett starkt redskap för att få mikroorganismer att framställametaboliter som de normalt inte producerar. Målet med detta projekt var rekombinantproduktion av gamma hydroxibutansyra (4-HB) genom att uttrycka två enzym i modellorganismen Escherichia coli. Dessa enzym bestod av α-ketoglutarat dekarboxylas (SucA) frånMycobacterium smegmatis samt 4-hydroxybutyrate dehydrogenas (4-HBd) från Clostridiumkluyveri. Resultaten visade att proteinerna lyckades utryckas i E. coli med en koncentration av0,9 g/L SucA och 9,8 g/L 4-HBd som uppnåddes efter rening. Utöver detta detekterades ävenviss enzymaktivitet genom att kopplad enzymreaktion mellan 4-HBd och SucA och mätakonsumtionen av NADH spektrofotometriskt över tid. Enzymen testades även försubstratspecificitet genom att köra reaktionen med substrat med olika längd på kolkedjan. Dåkunde en minskning i NADH koncentrationen ses men det gjordes det även för de negativakontrollerna vilket indikerar nedbrytning av NADH och inte konsumtion av NADH. Ingaslutsatser angående enzymens substratspecificitet kunde därför dras. Det sista som gjordes varatt sätta in båda generna i ett en plasmidsystem där båda generna sattes in på samma plasmid(pCDF duet) och uttrycktes framgångsrikt i E. coli Bl21DE3.

A quitosana é um polímero natural obtido a partir da desacetilação química da quitina, sendo a quitina o segundo polissacarídeo mais abundante na natureza. O interesse em pesquisas por novas aplicações da quitosana vem aumentando muito em diversas áreas, como na indústria farmacêutica, na indústria de cosméticos e de alimentos. Isso se deve às importantes características biológicas e físico-químicas inerentes à quitosana, como: biocompatibilidade, biodegradabilidade, propriedade de formação de filmes e fibras, complexação de metais e distintas atividades biológicas. Além disso, a presença de grupos amino na molécula da quitosana permite modificações químicas das mais diversas. No entanto, esta funcionalidade tem mostrado ser dependente, não apenas da sua estrutura química, mas também do seu tamanho molecular. Pois muitas das propriedades físico-químicas, e funcionais de uma cadeia polimérica são definidas pela sua massa molecular. O presente trabalho objetivou a síntese, caracterização e estudo da atividade antibacteriana de derivados de quitosana: quitosanas de baixa massa molecular, utilizando-se o peróxido de hidrogênio como agente oxidante; N-succinilquitosana e 2-carboxibenzamido-quitosana, de alta e baixa massa molecular, em presença de anidridos cíclicos, anidrido succínico e anidrido ftálico, respectivamente. Foram realizadas análises de caracterização dos derivados sintetizados por espectroscopia Raman e Infravermelho e avaliação de propriedades físico-químicas, como viscosidade e solubilidade. A efetividade antimicrobiana da quitosana e de seus derivados sintetizados, de alta e baixa massa molecular, foi avaliada frente à concentração final de 106UFC/mL de Escherichia coli através da determinação da concentração mínima inibitória (CMI). Os derivados de baixa massa molecular se mostraram mais solúveis e com menor viscosidade em relação às amostras de alta massa molecular. Em relação à atividade antibacteriana, nenhuma das amostras testadas exibiu ação antibacteriana significativa. / Chitosan is a natural polymer derived from the chemical deacetylation of chitin. Chitin is the second most abundant polysaccharide in nature. The interest in research for new applications of chitosan has been increasing fast in many areas, such as pharmaceuticals, cosmetics and foods industries. This is due to important biological and physical-chemical properties inherent to chitosan, such as biocompatibility, biodegradability, film-forming properties and fiber, metal complexation and distinct biological activities. Moreover, the presence of amino groups in the molecule of chitosan allows a wide range of chemical modifications. However, this feature has shown to be dependent not only on its chemical structure but also on their molecular size. Because many of the physico-chemical and functional characteristics of a polymer chain are defined by their molecular weight. This paper aims at the synthesis, characterization and study of antibacterial activity of chitosan derivatives, low molecular weight chitosan, using hydrogen peroxide as an oxidizing agent, N-succinylchitosan and 2-carboxybenzamido-chitosan, high and low molecular weight in the presence of cyclic anhydrides, succinic anhydride and phthalic anhydride, respectively. Analyses were performed to characterize the derivatives synthesized by Raman and Infrared spectroscopy and assessment of physicochemical properties such as viscosity and solubility. The antimicrobial effectiveness of chitosan and its derivatives synthesized, high and low molecular weight, was evaluated against the final concentration of Escherichia coli 106 UFC/mL by determining the minimum inhibitory concentration (MIC). The low molecular weight derivatives were more soluble and lessviscous for samples of high molecular weight. Regarding the antibacterial activity, none of the samples tested exhibited significant antibacterial activity.

A quitosana é um polímero natural obtido a partir da desacetilação química da quitina, sendo a quitina o segundo polissacarídeo mais abundante na natureza. O interesse em pesquisas por novas aplicações da quitosana vem aumentando muito em diversas áreas, como na indústria farmacêutica, na indústria de cosméticos e de alimentos. Isso se deve às importantes características biológicas e físico-químicas inerentes à quitosana, como: biocompatibilidade, biodegradabilidade, propriedade de formação de filmes e fibras, complexação de metais e distintas atividades biológicas. Além disso, a presença de grupos amino na molécula da quitosana permite modificações químicas das mais diversas. No entanto, esta funcionalidade tem mostrado ser dependente, não apenas da sua estrutura química, mas também do seu tamanho molecular. Pois muitas das propriedades físico-químicas, e funcionais de uma cadeia polimérica são definidas pela sua massa molecular. O presente trabalho objetivou a síntese, caracterização e estudo da atividade antibacteriana de derivados de quitosana: quitosanas de baixa massa molecular, utilizando-se o peróxido de hidrogênio como agente oxidante; N-succinilquitosana e 2-carboxibenzamido-quitosana, de alta e baixa massa molecular, em presença de anidridos cíclicos, anidrido succínico e anidrido ftálico, respectivamente. Foram realizadas análises de caracterização dos derivados sintetizados por espectroscopia Raman e Infravermelho e avaliação de propriedades físico-químicas, como viscosidade e solubilidade. A efetividade antimicrobiana da quitosana e de seus derivados sintetizados, de alta e baixa massa molecular, foi avaliada frente à concentração final de 106UFC/mL de Escherichia coli através da determinação da concentração mínima inibitória (CMI). Os derivados de baixa massa molecular se mostraram mais solúveis e com menor viscosidade em relação às amostras de alta massa molecular. Em relação à atividade antibacteriana, nenhuma das amostras testadas exibiu ação antibacteriana significativa. / Chitosan is a natural polymer derived from the chemical deacetylation of chitin. Chitin is the second most abundant polysaccharide in nature. The interest in research for new applications of chitosan has been increasing fast in many areas, such as pharmaceuticals, cosmetics and foods industries. This is due to important biological and physical-chemical properties inherent to chitosan, such as biocompatibility, biodegradability, film-forming properties and fiber, metal complexation and distinct biological activities. Moreover, the presence of amino groups in the molecule of chitosan allows a wide range of chemical modifications. However, this feature has shown to be dependent not only on its chemical structure but also on their molecular size. Because many of the physico-chemical and functional characteristics of a polymer chain are defined by their molecular weight. This paper aims at the synthesis, characterization and study of antibacterial activity of chitosan derivatives, low molecular weight chitosan, using hydrogen peroxide as an oxidizing agent, N-succinylchitosan and 2-carboxybenzamido-chitosan, high and low molecular weight in the presence of cyclic anhydrides, succinic anhydride and phthalic anhydride, respectively. Analyses were performed to characterize the derivatives synthesized by Raman and Infrared spectroscopy and assessment of physicochemical properties such as viscosity and solubility. The antimicrobial effectiveness of chitosan and its derivatives synthesized, high and low molecular weight, was evaluated against the final concentration of Escherichia coli 106 UFC/mL by determining the minimum inhibitory concentration (MIC). The low molecular weight derivatives were more soluble and lessviscous for samples of high molecular weight. Regarding the antibacterial activity, none of the samples tested exhibited significant antibacterial activity.

L’industrie textile utilise les microcapsules depuis de nombreuses années et beaucoup d’applications se sont développées notamment dans le domaine des cosmétotextiles. Les microcapsules utilisées pour ces applications sont souvent obtenues par polycondensation in situ du formaldéhyde et de la mélamine. La membrane réticulée assure une bonne tenue thermique et mécanique, indispensable au traitement appliqué lors du dépôt des microcapsules et de leur accroche sur textile. La présence résiduelle de formaldéhyde, classé cancérogène de catégorie 3, pose problème et peut être contrôlée par un post-traitement. Cependant, REACH va probablement diminuer les concentrations en formaldéhydes admises. Notre étude s’inscrit dans ce contexte, afin de développer des microcapsules sans formaldéhydes, à partir de monomères 100% d’origine naturelle selon des procédés respectueux de l’environnement. La membrane des microcapsules, synthétisée par polycondensation interfaciale entre le chlorure de succinyle et le 1,4 diaminobutane, donne lieu à la formation d’oligomères de polyamide 4,4 difficiles à caractériser car jamais étudié en détail. Un travail préliminaire de synthèse et de caractérisation de modèles a donc été effectué afin de nous aider dans la caractérisation de la membrane des microcapsules. Dans un second temps, nous avons optimisé les conditions opératoires de formation des microcapsules afin qu’elles répondent au cahier des charges. Les microcapsules obtenues et leur membrane polyamide 4,4 ont ensuite été caractérisées. Enfin, nous avons testé l’application de ces microcapsules sur textiles et étudié la résistance des cosméto-textiles obtenus aux frottements et au lavage en machine. / Textile industry has been using microcapsules for many years especially in the design of cosmeto-textiles. Microcapsules used for textile applications are often obtained by in situ polycondensation of formaldehyde and melamine. The crosslinked membrane ensures good thermal and mechanical properties, essential for treatment applied during microcapsules deposit and for their attachment onto textile. The residual presence of formaldehyde, classified as carcinogenic substance of level 3, is problematic and can be controlled by a post treatment. However, REACH directive will probably lower allowed formaldehyde concentrations. In this context, the aim of this work is to develop microcapsules without formaldehyde, from 100% bio-based monomers and using environmental friendly processes. Membrane microcapsules being synthesized by interfacial polycondensation between succinyl chloride and 1, 4-diaminobutane, this reaction results in polyamide 4 4 oligomers’ formation, difficult to characterize because this polymer has not yet been studied in detail. A preliminary work of syntheses and models characterizations has been performed to help the microcapsules membrane characterization. In a second step, we optimized the operating conditions of microcapsules formation to meet the specifications. The obtained microcapsules and their polyamide 4, 4 membrane were further characterized. Finally, we tested the microcapsules application onto textile and studied the obtained cosmeto-textile resistance to friction and washing machine.

ABSTRACT The porphyrias are a diverse group of metabolic disorders arising from diminished activity of enzymes in the heme biosynthetic pathway. They can present with acute neurovisceral symptoms, cutaneous symptoms, or both. The complexity of these disorders is demonstrated by the fact that some acute porphyria patients with the underlying genetic defect(s) are latent and asymptomatic while others present with severe symptoms. This indicates that there is at least one other risk factor required in addition to the genetic defect for symptom manifestation. A systematic review of the heme biosynthetic pathway highlighted the involvement of a number of micronutrient cofactors. An exhaustive review of the medical literature uncovered numerous reports of micronutrient deficiencies in the porphyrias as well as successful case reports of treatments with micronutrients. Many micronutrient deficiencies present with symptoms similar to those in porphyria, in particular vitamin B6. It is hypothesized that a vitamin B6 deficiency and related micronutrient deficiencies may play a major role in the pathogenesis of the acute porphyrias. In order to further investigate the porphyrias, a computational model of the heme biosynthetic pathway was developed based on kinetic parameters derived from a careful analysis of the literature. This model demonstrated aspects of normal heme biosynthesis and illustrated some of the disordered biochemistry of acute intermittent porphyria (AIP). The testing of this model highlighted the modifications necessary to develop a more comprehensive model with the potential to investigated hypotheses of the disordered biochemistry of the porphyrias as well as the discovery of new methods of treatment and symptom control. It is concluded that vitamin B6 deficiency might be the risk factor necessary in conjunction with the genetic defect to trigger porphyria symptoms.