<p>The successful genomic era has resulted in a great demand for efficient production and purification of proteins. The main objective of the work described in this thesis was to develop methods to facilitate recovery of target proteins after recombinant production in Escherichia coli.</p><p>A positively charged purification tag, Z_basic, has previously been constructed by protein design of a compact three-helix bundle domain, Z. The charged domain was investigated for general use as a fusion partner. All target proteins investigated could be selectively captured by ion-exchange chromatography under conditions excluding adsorption of the majority of Escherichia coli host proteins. A single cation-exchange chromatography step at physiological pH was sufficient to provide Z_basic fusion proteins of high purity close to homogeneity. Moreover, efficient isolation directly from unclarified <i>Escherichia coli</i> homogenates could also be accomplished using an expanded bed mode. Since the intended use of a recombinant protein sometimes requires removal of the purification tag, a strategy for efficient release of the Z_basic moiety using an immobilised protease was developed. The protease columns were reusable without any measurable decrease in activity. Moreover, subsequent removal of the released tag, Z_basic, was effected by adsorption to a second cation-exchanger. </p><p>Using a similar strategy, a purification tag with a negatively charged surface, denoted Z_acid, was constructed and thoroughly characterised. Contrary to Z_basic, the negatively charged Z_acid was highly unstructured in a low conductivity environment. Despite this, all Z_acid fusion proteins investigated could be efficiently purified from whole cell lysates using anion-exchange chromatography</p><p>Synthesis of polypeptides occurs readily in Escherichia coli providing large amounts of protein in cells of this type, albeit often one finds the recombinant proteins sequestered in inclusion bodies. Therefore, a high throughput method for screening of protein expression was developed. Levels of both soluble and precipitated protein could simultaneously be assessed <i>in vivo</i> by the use of a flow cytometer. </p><p>The positively charged domain, Z_basic, was shown also to be selective under denaturing conditions, providing the possibility to purify proteins solubilised from inclusion bodies. Finally, a flexible process for solid-phase refolding was developed, using Z_basic as a reversible linker to the cation-exchanger resin.</p>

Les lysophosphatidylcholines (LPCs), molécules amphiphiles, peuvent jouer le rôle d'un vecteur pour des acides gras d'intérêt. Ces molécules peuvent être synthétisées par voie enzymatique. Ce travail a pour objectif de synthétiser des LPCs contenants des acides gras mono ou poly insaturés par voie enzymatique. La sn-3-glycerophosphatidylcholine (GPC), l'acide oléique (C18 : 1, ω9), l'acide linoléique (C18 : 2, ω6) et l'acide docosahexènoïque (C22 : 6, ω3) ont été utilisés comme substrats. Ces synthèses ont été réalisées par estérification sans solvant en utilisant une lipase immobilisée. Du point de vue cinétique, une optimisation des conditions de la synthèse de l'oléoyl-LPC a été effectuée en étudiant chaque facteur d'une façon indépendante. Les conditions optimales sont : Lipozyme RM-IM (10% par rapport la masse totale des substrats), un rapport molaire de 20 (acide oléique /GPC), ni ajout ni élimination d'eau au milieu réactionnel, une température de 50°C et une agitation à 750rpm. Ces conditions permettent d'avoir 75% d'oléoyl-LPC en 24h. L'oléoyl-LPC a été purifiée sur une cartouche de silice et caractérisée par SM pour confirmer son identité. L'optimisation de la synthèse de linoléoyl-LPC a été faite via un plan d'expériences. C'est une approche qui permet de varier simultanément les paramètres étudiés. Parmi les 17 combinaisons étudiées, une combinaison a permis d'obtenir 93% de linoléoyl-LPC en 6h. Ce résultat a été confirmé en répétant la manipulation 4 fois. Comme l'oleoyl-LPC, la linoleoyl-LPC a été purifiée et caractérisée par SM. Des essais préliminaires ont montré que la synthèse de DHA-LPC est faisable. 30% de DHA-LPC a été obtenu au bout de 72h de réaction. Enfin, il faut noter que la quantité produite de di-acyl-PC était toujours faible par rapport à celle de l'acyl-LPC. Ce dernier résultat est expliqué par une apparition faible du phénomène de la migration du groupement acyle. / The Lysophosphatidylcholines (LPCs), amphiphilic molecules, can act as a carrier for fatty acids of interest. These molecules can be synthesized enzymatically. This work aims to synthesize LPCs containing mono or poly unsaturated fatty acids enzymatically. Sn-3-glycerophosphatidylcholine (GPC), oleic acid (C18: 1, 9ω), linoleic acid (C18: 2, 6ω) and docosahexaenoic acid (C22: 6, 3ω) were used as substrates. These syntheses were performed by esterification without solvent using an immobilized lipase. From kinetic point of view, optimization of conditions for synthesis of oleoyl-LPC was carried out by studying each factor independently. The obtained optimal conditions are : Lipozyme RM-IM (10% w/w), a molar ratio of 20 (oleic acid / GPC), neither addition or elimination of water was applied to the reaction mixture, a temperature at 50° C and stirring at 750rpm. These conditions allow to obtain 75% oleoyl-LPC within 24 hours. The oleoyl-LPC was purified on a silica cartridge and analyzed by MS to confirm its identity. The optimization of linoleoyl-LPC synthesis was made using a design of experiments. This is an approach that can simultaneously vary the parameters studied. Among the 17 combinations studied, one combination allows to obtain 93% of linoleoyl-LPC in 6 hours. This result was confirmed by repeating this manipulation 4 times. Like oleoyl-LPC linoleoyl-LPC was purified and characterized by MS. Preliminary trials have shown that the syntheis of DHA-LPC is feasible. 30% of DHA-LPC was obtained after 72h of reaction. Finally, it should be noted that the produced amount of di-acyl-PC is always low compared to that of the acyl-LPC. This latter is explained by a low appearance of the phenomenon of acyl migration.

Investments in carbon capture from industrial emissions have been on the rise in recent years, having reached over $200 million in 2021 as compared to 2015’s $13 million. The Paris Agreement, signed by 196 parties globally in 2015, is purported to be the primary driver for this, with its ambitious goal of limiting global surface temperature rise to 1.5°C by the year 2100 as compared to the pre-industrial era. Achievement of a carbon-neutral future for industries has been sought by experts in more than a few ways, which include attempts directed towards re-designing current manufacturing processes to produce inherently low CO2 emissions. Although eventual elimination of carbon emissions forms the ultimate goal, complete avoidance of CO2 production does not seem probable for all industrial sectors. Emissions from industries in the medium to long term are thus foreseen to be composed between 0.5% and 7% of CO2 by moles (roughly between 1% and 10% by mass), depending on the level of dilution occurring during the various flue gas treatment procedures between their source and the capture unit. An assessment of the capabilities of two popular and one prospective carbon capture technologies in capturing CO2 from such emissions of the future has been made in this work to aid investors make informed decisions about a suitable technology. The monoethanolamine-based (MEA) absorption system, one of the most popular choices today, was found to be well capable of treating emissions composed of CO2 in proportions as low as 0.6% by mole (or ∼1% by mass) with capture rates well over 95%. Its thermal energy intensity ranged between 3.59 MJth/kgCO2 captured and 10.23 MJth/kgCO2 captured with an associated levelised cost of capture ranging between €20.36/tonneCO2 captured and €141.97/tonneCO2 captured going from the 10% concentrated to the 1% concentrated stream by mass. In comparison, the benfield system was found to effect much lower CO2 capture rates ranging between 35% and 88%, making it unsuitable for treatment of low CO2 concentrated streams. Even with such poor performance at high pressures of operation, its energy demand ranged between 3.9 MJth/kgCO2 captured and 11.07 MJth/kgCO2 captured with an associated levelised cost of capture between €174.28/tonneCO2 captured and €4209.06/tonneCO2 captured. The immobilised amine-based system, in what is considered to be a non-optimised configuration yet, was found to capture nearly 100% of the entering CO2 with energy consumption ranging between 3.71MJth/kgCO2 captured and 11.8 MJth/kgCO2 captured for extremely high, but improvable levelised costs of capture ranging between €674.31/tonneCO2 captured and €3488.42/tonneCO2 captured. Exhibiting comparable energy performance to the mature MEA-based absorption system’s even in its non-optimised configuration, the immobilised amine-based adsorption system was found to possess potential to be the carbon capture technology of the future for treatment of low CO2-concentrated effluent streams. / Investeringar i koldioxidavskiljning från industriella utsläpp har ökat de senaste åren och nått över 200 miljoner USD 2021 jämfört med 2015 års 13 miljoner USD. Parisavtalet, som undertecknades av 196 parter globalt 2015, påstås vara den främsta drivkraften för detta, med det ambitiösa målet att begränsa den globala yttemperaturökningen till 1,5°C till år 2100 jämfört med den förindustriella eran. Att uppnå en koldioxidneutral framtid för industrier har eftersträvats av experter på mer än ett fåtal sätt, vilket inkluderar försök inriktade på att omdesigna nuvarande tillverkningsprocesser för att producera låga CO2-utsläpp. Även om fullständig eliminering av koldioxidutsläpp utgör det ideala målet, är det inte troligt att CO2-produktion kan undvikas helt för att alla industrisektorer. Utsläppen från industrier på medellång till lång sikt förväntas därför utgöra mellan 0,5 % och 7 % av CO2 per mol (ungefär mellan 1 % och 10 % i massa), beroende på nivån av utspädning som inträffar under de olika rökgasbehandlingsprocedurerna mellan utsläppskällan och fångstenheten. I det här arbetet har två traditionella och en potentiellt blivande koldioxidavskiljningsteknik jämförts och en bedömning av deras förmåga att fånga in CO2 från framtida utsläpp har gjorts i syfte att hjälpa investerare att göra ett klokt val. Det monoetanolaminbaserade (MEA) absorptionssystemet, ett av de mest populära valen idag, visade sig vara väl kapabelt att behandla utsläpp med CO2-koncentrationer så låga som 0,6 molprocent (eller 1 massprocent) med fångsthastigheter långt över 95 %. Dess termiska energiintensitet varierade mellan 3,59 MJth/kgCO2 captured och 10,23 MJth/kgCO2 captured med en tillhörande utjämnad kostnad för fångst mellan €20,36/tonCO2 captured och €141,97/tonCO2 captured från 10 % koncentrerad till 1 % koncentrerad ström i massa. Som jämförelse visade sig benfield-systemet ge mycket lägre CO2-avskiljningshastigheter på mellan 35 % och 88 %, vilket gör metodenolämplig för behandling av gasströmmar med låg CO2-koncentration. Den dåliga prestandan uppvisades trots höga drifttryck och metoden medförde en energiintensitet mellan 3,9MJth/kgCO2 captured till 11,07MJth/kgCO2 captured samt en snittkostnad mellan €174/tonCO2 captured till €4209,06/tonCO2 captured. Det immobiliserade aminbaserade systemet anses vara en icke-optimerad konfiguration men visade sig trots det fånga upp nästan 100 % av inkommande CO2 med en energiförbrukning på mellan 3,71 MJth/kgCO2 captured och 11,8 MJth/kgCO2 captured. De extremt höga, men dock förbättringsbara, snittkostnaderna för infångningen sträcker sig mellan €674/tonCO2 captured och €3488,42/tonCO2 captured. Det immobiliserade aminbaserade adsorptionssystemet uppvisar jämförbar energiprestanda som det mogna MEA-baserade absorptionssystemet även i sin icke-optimerade konfiguration.

Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2009. / Bibliography: p. 200-212. / Introduction -- Shotgun proteomic analysis of rat liver membrane proteins -- A combination of immobilised pH gradients improve membrane proteomics -- Affects of tumor-induced inflammation on membrane proteins abundance in the mouse liver -- Affects of tumor-induced inflammation on biochemical pathways in the mouse liver -- General discussion -- References. / The aim of this thesis was to develop a proteomics methodology that improves the identification of membrane proteomes from mammalian liver. Shotgun proteomics is a method that allows the analysis of proteins from cells, tissues and organs and provides comprehensive characterisation of proteomes of interest. The method developed in this thesis uses separation of peptides from trypsin digested membrane proteins by immobilised pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two dimensional shotgun proteomics. In this thesis, peptide IPG-IEF was shown to be a highly reproducible, high resolution analytical separation that provided the identification of over 4,000 individual protein identifications from rat liver membrane samples. Furthermore, this shotgun proteomics strategy provided the identification of approximately 1,100 integral membrane proteins from the rat liver. The advantages of using peptide IPG-IEF as a shotgun proteomics separation dimension in conjunction with label-free quantification was applied to a biological question: namely, does the presence of a spatially unrelated benign tumor affect the abundance of mouse liver proteins. IPG-IEF shotgun proteomics provided comprehensive coverage of the mouse liver membrane proteome with 1,569 quantified proteins. In addition, the presence of an Englebreth-Holm-Swarm sarcoma induced changes in abundance of proteins in the mouse liver, including many integral membrane proteins. Changes in the abundance of liver proteins was observed in key liver metabolic processes such as fatty acid metabolism, fatty acid transport, xenobiotic metabolism and clearance. These results provide compelling evidence that the developed shotgun proteomics methodology allows for the comprehensive analysis of mammalian liver membrane proteins and detailed some of the underlying changes in liver metabolism induced by the presence of a tumor. This model may reflect changes that could occur in the livers of cancer patients and has implications for drug treatments. / Mode of access: World Wide Web. / 609 p. ill. (some col.)

This work aims at modelling and evaluating a new type of photocatalytic reactors, named fixed-bed photocatalytic membrane reactor (FPMR). Such reactors are based on the deposition of a thin layer of photocatalysts on a permeable substrate by filtration. This layer serves as a photocatalytic membrane, named fixed-bed photocatalytic mem-brane (FPM), which is perpendicularly passed by the reactant solution and illuminated by a suitable light source. One advantage of FPMs is their renewability. The model, which was developed for this reactor, relates the overall reaction rate in the FPM with the intrinsic reaction kinetic at the catalyst surface and accounts for light intensity, structural and optical layer properties as well as the mass transfer in the pores. The concept of FPMR was realised by using a flat sheet membrane cell. It facilitated principal investigations into the reactor performance and the validity of the model. For this purpose, the photocatalytic degradation of organic compounds, such as meth-ylene blue and diclofenac sodium, was conducted at varying conditions. Pyrogenic ti-tania was used as a photocatalyst. The experimental data support the developed mod-el. They also indicate a significant impact of the flow conditions on the overall photo-catalytic activity, even though the Reynolds number in the FPM was very small; the to-tal mass transfer rate in the FPM amounted to more than 1.0 s−1. The experiments also showed a sufficient structural strength of the FPM and photocatalytic stability. In addi-tion, the renewal and regeneration of FPMs was successfully demonstrated. Furthermore, another FPMR was designed by means of submerged ceramic mem-branes. This reactor was mainly used to assess the effectiveness and efficiency of FPMRs at the example of the photocatalytic degradation of oxalic acid. The correspond-ing reactor was run closed loop and in continuous mode. The effectiveness of the reac-tor was evaluated based on common descriptors, such as apparent quantum yield, photocatalytic space-time yield and light energy consumption. The results showed that the FPMR based on submerged ceramic membrane had a higher efficiency than other reported photocatalytic reactors. The comparison of the different modes of operation revealed that the closed loop FPMR is most efficient with regard to light energy con-sumption. Finally, this work discusses the up-scaling of FPMRs for industrial applications and proposes a solution, which can e.g. be employed for wastewater treatment or CO2 conversion.:Abstract iii Kurzfassung v Acknowledgment vii Contents ix Nomenclature xiii 1 Introduction 1 1.1 Motivation 1 1.2 Aim and objectives of the work 3 1.3 Thesis outline 3 2 Heterogeneous photocatalytic reactors 5 2.1 Introduction to photocatalysis 5 2.2 Processes in heterogeneous photocatalysis 6 2.2.1 Optical phenomena 7 2.2.2 Mass transfer 8 2.2.3 Adsorption and desorption 9 2.2.4 Photocatalytic reactions 10 2.2.5 Factors affecting heterogeneous photocatalysis 12 2.3 Photocatalytic reactor systems towards water treatment 16 2.3.1 Introduction to photocatalytic reactors 16 2.3.2 Development of photocatalytic reactor designs 17 2.3.3 Quantitative criteria for evaluating photocatalytic reactor designs 21 2.4 Cake layer formation in membrane microfiltration 22 2.4.1 Suspension preparation 22 2.4.2 Cake layer formation 23 2.5 Fluid flow through a fixed bed of particles 25 2.5.1 Pressure drop through a fixed-bed 25 2.5.2 Liquid-solid mass transfer correlation in fixed-bed 25 3 Concept and mathematical modelling of FPMRs 29 3.1 Concept of fixed-bed photocatalytic membrane reactors 29 3.2 Modelling of fixed-bed photocatalytic membrane reactors 31 3.3 Model sensitivity analysis 37 3.4 Chapter summary 39 4 FPMR realised with flat sheet polymeric membrane 41 4.1 Introduction 41 4.2 Materials and set-up 41 4.2.1 Materials 41 4.2.2 Experimental set-up 43 4.3 Experiments and methods 48 4.3.1 Formation of fixed-bed photocatalytic membrane 48 4.3.2 Reactor performance 50 4.3.3 Parameters study and model verification 53 4.3.4 Catalyst layer characterisation 56 4.3.5 Measurement and evaluation of photocatalytic activity of FPM 59 4.4 Results and model verification 60 4.4.1 Reactor performance 60 4.4.2 Influence parameters 71 4.4.3 Model verification 79 5 FPMR realised with submerged ceramic membrane 92 5.1 Introduction 92 5.2 Materials and reactor set-up 93 5.2.1 Reactor set-up 93 5.2.2 Chemicals 97 5.3 Experiments and methods 97 5.3.1 Formation of fixed-bed photocatalytic membranes 97 5.3.2 Photocatalytic performance 97 5.3.3 Parameter study 98 5.3.4 Reactor model for calculating reaction rate constant of FPM 99 5.3.5 Comparison of different reactor schemes 102 5.4 Results and discussions 105 5.4.1 Reactor performance 105 5.4.2 Consistency of CPMR and LPMR data 107 5.4.3 Influence of catalyst loading 108 5.4.4 Influence of permeate flux and light intensity 109 5.4.5 Reactor efficiency 111 5.4.6 Comparison of different reactor schemes 113 5.5 Proposed up-scaled FPMR systems 113 5.6 Concluding remarks 116 6 Conclusion and outlook 118 6.1 Summary of thesis contributions 118 6.2 Discussion and outlook 120 References 122 List of Figures 134 List of Tables 138 Appendix A Calibration 139 A.1 Distribution of light intensity on the surface of catalyst layer 139 A.2 Concentration and absorbance of diclofenac 141 A.3 TOC concentration and electrical conductivity of oxalic acid 141 A.4 Concentration and absorbance of methylene blue 142 Appendix B Mathematical modelling 143 B.1 Influence of axial dispersion on the reaction rate 143 B.2 Special case 146 Appendix C Comparison the photocatalytic activity of TiO2 and ZnO 147 Appendix D Mathematical validation of model for LPMR and CPMR 148 D.1 Model for LPMR (cf. Eq. (5 12)):148 D.2 Model for CPMR (cf. Eq. (5 17)) 149 Appendix E Particle size distribution 151