Thesis (MScEng) -- University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The grape juice concentrate (GJC) plant of the KWV at Robertson spent significant amounts of money on the decolourisation of grape juice concentrate. A chemically activated powdered activated carbon (PAC) purchased from Norit, namely CA1, was used as decolourisation product. Apart from the expenses involved, it contributed largely to the solid waste produced at this plant. A way was sought to minimise or prevent GJC discolourisation (and possibly solid waste) without increasing operating expenses. Browning reactions in GJC are as old as the product itself. Numerous researchers have studied the origins of these reactions, the reactants and products involved, as well as the reaction kinetics of these reactions. From the work of these researchers four possible browning reaction pathways were identified, namely: • enzymatic oxidative browning, • non-enzymatic oxidative browning, • non-enzymatic browning (the Maillard reaction), and • caramelisation. It was also identified that 5-hydroxymethylfurfural (HMF) are indicative of the browning potential of GJC. A method to analyse for HMF (quantitative and qualitative) was develop for the purposes of this study, namely positive electron-spray ionisation preceded by high-pressure liquid chromatography (HPLC) and followed by dual mass spectrometry. This method showed good repeatability and was used to analyse all samples generated during this study. It was confirmed that the manufacturing process at this plant favours nonenzymatic browning reactions, since mild heat treatment deactivates enzymes. Further investigation indicated that the overruling browning reaction on this plant was non-enzymatic oxidative browning. It was shown that neither the presence, nor the absence of protein had any effect on the rate of formation of HMF. It was, however, confirmed that HMF formation could be attributed to high temperatures and prolonged exposure to these temperatures. Other adsorption products were evaluated against the then current PAC (CA1), namely a steam activated PAC supplied by Norit, SA4, and a polymeric adsorbent, Polyclar V (polyvinylpolypyrrolidone/PVPP). Both SA4 and PVPP indicated superior HMF adsorption capacities. Replacing CA1 with SA4 could result in operating expenses savings and possible solid waste reduction. However, PVPP were too expensive to be considered an economically viable replacement for CA1. Improved concentration technologies such as reverse osmosis (RO) membrane concentration followed by centrifugal evaporation (CE) or twostage CE should be considered as possible replacement for the existing concentration technology (multi-stage falling film evaporator). This should decrease heat treatment/exposure by more than 90% and thus reduce browning significantly. An added advantage could be the reduction of solid waste, since less (if not no) decolourisation will be required. Alternatively, juice should be stored with added sulphur dioxide (SO2), since it was shown that this juice contained much lower HMF concentrations than diluted concentrate (stored for the same time). This should reduce heat exposure by up to 50% and thus minimise browning reactions. / AFRIKAANSE OPSOMMING: Die druiwesapkonsentraat (DSK) aanleg van die KWV in Robertson het jaarliks aansienlike bedrae geld spandeer tydens die ontkleuringsproses van DSK. ‘n Chemies geaktiveerde verpoeierde koolstof (GVK) verkrygbaar van Norit, naamlik CA1, is gebruik as ontkleuringsproduk. Buiten die kostes verbonde aan hierdie produk het dit ook grootliks bygedra tot soliede afval by hierdie aanleg. Oplossings is gesoek om die verbruining/ontkleuring van DSK (en dalk ook soliede afval) te verminder (of selfs te voorkom) sonder om bedryfskostes te verhoog. Verbruiningsreaksies in DSK bestaan al so lank soos DSK self. Verskeie navorsers het die oorsake, reaktante, produkte en reaksiekinetika van hierdie reaksies oor die jare heen bestudeer. Uit die werk van sommige van hierdie navorsers kon vier moontlike verbruiningsreaksieroetes geïdentifiseer word, naamlik: • ensiematiese oksidatiewe verbruining, • nie-ensiematiese oksidatiewe verbruining, • nie-ensiematiese verbruining (die Maillard-reaksie), en • karamelisering. Daar was verder geïdentifiseer dat 5-hidroksiemetielfurfuraal (HMF) aanduidend is van die verbruiningspotensiaal van DSK. ‘n Analitiese metode (kwalitatief en kwantitatief) om vir HMF te analiseer is vir die doel van hierdie studie ontwikkel, naamlik positiewe elektronsproei ionisasie, voorafgegaan deur hoëdruk vloeistof chromatografie en gevolg deur dubbele massa spektrometrie. Hierdie analitiese metode het goeie herhaalbaarheid getoon en was deurgaans gebruik om monsters te analiseer gedurende hierdie studie. Dit was bevestig dat die vervaardigingsproses by hierdie aanleg nieensiematiese verbruiningsreaksies begunstig, aangesien geredelike hittebehandeling ensieme deaktiveer. Verdere navorsing het getoon dat die oorheersende verbruiningsreaksies by hierdie aanleg nie-ensiematiese oksidatief van aard is. Resultate het getoon dat proteinstabiliteit geen invloed op die vormingstempo van HMF het nie. Dit was egter bevestig dat vorming van HMF direk verband hou met hoë temperature en lang blootstellingsperiodes aan hierdie temperature. Ander adsorpsieprodukte was vergelyk met die huidige GVK (CA1), naamlik ‘n stoom geaktiveerde verpoeierde koolstof (Norit se SA4) en ‘n polimeriese adsorbant, Polyclar V (polivinielpolipirrolidoon/PVPP). Beide SA4 en PVPP het CA1 oortref wat betref HMF adsorpsie. Moontlike bedryfskostebesparings (en soliede afval verminderings) potensiaal bestaan indien CA1 vervang word met SA4. Die teenoorgestelde is egter waar vir PVPP wat bedryfskoste aangaan. Instede van die huidige verdampinstegnologie, naamlik vallendefilmverdamping, hoort verbeterde konsentrasietegnologieë soos tru-osmose membraankonsentrasie gevolg deur sentrifugale verdamping, of, alternatiewelik, twee-stadium sentrifugale verdamping, orrweeg te word. Op hierdie wyse behoort hittebehandeling (en dus verbruining) met sowat 90% verminder te word. ‘n Moonlike addisionele voordeel is die vermindering van soliede afval aangesien minder ontkleuring nodig sal wees. Indien die verbeterde tegnologieë te duur is moet daar gekyk word daarna om die ongekonsentreerde sap met addisionele swaweldioksied (SO2) te stoor, aangesien veel laer HMF konsentrasies in sulke sap waargeneem is as in verdunde direkte konsentraat wat vir dieselfde typerk gestoor is. Hittebehandeling sal op hierdie wyse met tot 50% verminder word (en dus verbruining ook).

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Maceration enzymes for the wine industry are preparations containing mainly pectinases, cellulases and hemicellulases, used during wine making to degrade the berry cell walls and release polyphenolic and aroma molecules to increase wine quality. These types of enzymes are also used for the harvesting of revenue-generating molecules from pomace (skins, pulp and seeds from grape processing waste), or as processing aids when used in the production of bioethanol. Grape berry cell walls are recalcitrant towards degradation, therefore knowledge about their structures and compositions, as well as how the application of enzymes modify these structures is essential in order to optimise these processes. The aim of this study was to extend current knowledge by using a mixture of existing and novel methodologies to study grape berry cell walls by focusing on the profiles of polymers present in the walls. Cell wall profiling techniques used in this study include the Comprehensive Microarray Polymer Profiling (CoMPP) method that employs monoclonal antibodies and Carbohydrate Binding Modules (CBM) which specifically recognise the polymers in the plant cell wall. With this method we measured the abundance of specific polymers and traced the fluctuation in their levels of abundance as influenced by external factors such as enzyme hydrolysis. The CoMPP method was coupled with monosaccharide profile analysis by GC-MS to determine the building blocks of the cell wall polymers, as well as with Infrared Spectroscopy to monitor the changes in the bulk chemistry profile. Data sets generated by the cell wall profiling methods were analysed with uni- and multivariate statistical methods to detect the major patterns in the data. This study highlighted the cell wall differences on the polymer level, in the berry skin cells of Pinotage grapes at different ripeness levels and how it changes during a standard wine fermentation, leading to the release of homogalacturonans and the exposing of arabinogalactan proteins. When maceration enzymes were added, further depectination was evident and the enzymes unravelled the cell wall of the ripe grapes. In overripe grapes no additional degradation could be observed due to maceration enzyme actions, presumably indicating that the endogenous grape enzymes already caused extensive degradation. When purified enzymes were incubated under buffered conditions with isolated skin cell walls from Pinotage grapes or with Chardonnay grape pomace, different levels of enzymatic hydrolysis were observed and defined. The sequence in which cell wall polymers were extracted, and the influence of specific enzymes in facilitating the extraction process, provided important information on the accessibility of specific cell wall polymers. Synergistic action between, for example an endo-polygalacturonase (EPG) and an endo-glucanase (EG) was demonstrated with CoMPP. This EPG and EG synergism was also demonstrated with a yeast strain (a Saccharomyces paradoxus x S. cerevisiae hybrid) fermented in a buffered pomace suspension. This yeast strain has a native EPG and was engineered to also express a recombinant EG from a genome integrated cassette. The cell walls isolated from the pomace after fermentation were unravelled and depectination took place, as evident from CoMPP data. The cell wall profiling techniques used in this study were proven to be fast and sensitive. It provided insights into the structure of grape cell walls and was used to evaluate the changes due to ripening, fermentation, enzymatic hydrolysis and a heat pre-processing treatment. In addition to the knowledge gained, we also demonstrated that these techniques can be used to evaluate different enzymes and enzyme combinations as well as the potential of microorganisms to degrade grape tissue. / AFRIKAANSE OPSOMMING: Maserasie ensieme vir die wynindustrie is ensiem mengsels wat hoofsaaklik pektinases, sellulases en hemisellulases bevat en word tydens wynbereiding gebruik om die druifkorrel se selwand af te breek, die polifenole en aroma molekules vry te stel en sodoende die wyn kwaliteit te verbeter. Hierdie soort ensieme word ook gebruik om inkomste-genererende molekules vanuit druiweprosesserings afval (doppe, pulp en pitte) te isoleer, en ook as prosesserings hulpmiddels in die produksie van bioetanol. Druifkorrel selwande is weerstandig teen ensiem afbraak en daarom is kennis oor die struktuur en samestelling van die selwand, asook hoe die selwand strukture deur die toediening van ensieme verander word noodsaaklik om sodoende hierdie prosesse te optimaliseer. Die doel van hierdie studie was om die huidige kennis uit te brei deur bestaande asook nuwe metodes te gebruik om die druifkorrel selwand te bestudeer met die fokus op die polimeerprofiel van die selwande. Selwand karakteriserings tegnieke wat in hierdie studie gebruik is sluit in die Comprehensive Microarray Polymer Profiling (CoMPP) metode wat monoklonale teenliggaampies en koolhidraat bindende modules (Carbohydrate binding modules, CBMs) wat spesifiek die selwandpolimere van die plant selwand herken, gebruik. Met hierdie metode het ons die vlakke van spesifieke polimere gemeet asook die skommeling in hulle vlakke soos dit beïnvloed is deur eksterne faktore soos ensiem hidroliese. Die CoMPP metode is tesame met monosakkaried profiel analise, met behulp van GC-MS, wat die boublokke van die selwand polimere bepaal, asook infrarooi spektroskopie om die veranderinge in die oorhoofse chemiese profiel te bepaal, gebruik. Datastelle wat met die selwand karakteriserings tegnieke gegenereer is, is ontleed met een- en multiveranderlike statistiese metodes om die hoof tendense in die data op te spoor. Hierdie studie het die selwand verskille, op die polimeervlak, van Pinotage druiwe uitgelig. Verskillende rypheidsgrade asook hoe dit verander tydens ‘n standaard wynfermentasie is gevolg. Laasgenoemde het die vrystelling van homogalakturonaan en die ontbloting van arabinogalaktoproteïene tot gevolg gehad. Met die byvoeging van maserasie ensieme was dit duidelik dat addisionele pektienverwydering plaasgevind het en dat die ensieme die selwand van die ryp druiwe ontrafel het. In oorryp druiwe was daar geen addisionele selwand afbreking sigbaar as gevolg van die aksie van maserasie ensieme nie, wat moontlik aandui dat die inherente druif ensieme reeds uitgebreide selwand afbraak versoorsaak het. Wanneer gesuiwerde ensieme met geïsoleerde selwande van Pinotage druiwedoppe en met Chardonnay druiweprosesserings afval geïnkubeer is onder gebufferde kondisies, is verskillende vlakke van ensiematiese hidroliese waargeneem en geklassifiseer. Die volgorde waarin die selwand polimere geëkstraheer is, asook die invloed van spesifieke ensieme in die bevordering van die ekstraksie proses, het belangrike inligting verskaf oor die toeganglikheid van spesifieke selwand polimere. Sinergistiese aksie tussen, byvoorbeeld ‘n endo-poligalakturonase (EPG) en ‘n endo-glukanase (EG) is geidentifiseer met behulp van die CoMPP data. Hierdie EPG en EG sinergisme is ook geïllustreer met ‘n gisras (‘n Saccharomyces paradoxus x S. cerevisiae hibried) wat in ‘n gebufferde druifprosesserings afval suspensie gefermenteer het. Hierdie gisras het ‘n endogene EPG en is ontwerp om ook ‘n rekombinante EG uit te druk vanaf ‘n genoom geïntegreerde kasset. Die selwande van die druiweprosesserings afval wat na die fermentasie geïsoleer is, was ontrafel en pektienverwydering het plaasgevind, soos bevestig met CoMPP data. In hierdie studie is bewys dat die selwand karakteriserings tegnieke vinnig en sensitief is. Dit het insigte verskaf oor die struktuur van die druifselwand en is gebruik om die veranderinge as gevolg van rypheidsverskille, wynfermentasie, ensiem hidroliese en hitte prosessering te evalueer. Buiten die bydraes tot kennis oor hierdie onderwerpe, is die bruikbaarheid van hierdie tegnieke ook aangetoon, veral in die evaluasie van verskillende ensieme en ensiemkombinasies, asook mikroörganismes vir die afbraak van druifweefsel.

In all sequenced genomes, a large fraction of predicted genes encodes proteins of unknown biochemical function and up to 15% of the genes with ‘‘known’’ function are mis-annotated. Several global approaches are being employed to predict function, including sequence similarity searches, analysis of gene expression, protein interaction, and protein structure. Enzymes comprise a group of target proteins that require experimental characterization for accurate functional annotations. Here I applied enzyme genomics to identify new enzymes by screening individually purified proteins for enzymatic activity under relaxed reaction conditions, which allowed me to identify the subclass or sub-subclasses of enzymes to which the unknown protein belongs. Further biochemical characterization of proteins was facilitated by the application of secondary screens with natural substrates (substrate profiling). Application of general enzymatic screens and substrate profiling greatly sped up the identification of biochemical function of unknown proteins and the experimental verification of functional predictions produced by other functional genomics approaches. As a test case, I used this approach to characterize the members of the haloacid dehalogenase (HAD)-like hydrolase superfamily, which consists mainly of uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Low sequence similarity between the members of the HAD superfamily precludes the computational prediction of their substrates and functions. Using a representative set of 80 phosphorylated substrates I characterized the phosphatase activities of 21 soluble HADs from Escherichia coli and seven soluble HADs from Saccharomyces cerevisiae. E. coli HADs show broad and overlapping substrate specificity against a wide range of phosphorylated metabolites. The yeast enzymes were more specific, and one protein also showed protein phosphatase activity. Comparison of HAD substrate profiles from two model organisms showed several “functional niches” that are occupied by HADs, which include hydrolysis of nucleotides, phosphoglycolate, phosphoserine, and pyridoxal phosphate. I proposed the cellular function for a number of HADs from both organisms based on substrate specificities. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, E. coli YniC.

The rehabilitation of degraded soils, such as those associated with post-mining sites, requires knowledge of the soil ecosystem and its physical, chemical, and biological composition in order for rehabilitation efforts to fulfil the long-term goal of reconstructing a stable ecosystem for rehabilitated mine soil. This study addresses the need for appropriate assessment criteria to determine the progress of rehabilitation and subsequently the success of management practices. Significant contributions made by this investigation included the establishment of minimum and maximum values for microbial community measurements from two case studies of rehabilitated coal discard sites. Furthermore, it was shown that there was no relationship between changes in microbial community function and structure and the rehabilitation age of the sites. Following this, the considerable impact of management practices on microbial communities was illustrated. The first part of the study investigated the temporal changes in microbial community function and structure in a chronosequence of rehabilitated coal discard sites aged 1 to 11 years. The most important observation made during the investigation of the microbial communities in the different aged soil covers of the rehabilitated coal discard sites, was that there was no relationship between rehabilitation age and microbial activity or abundance of certain microbial groups. What was responsible for a clear differentiation between sites and a shift in microbial community attributes was the management practices applied. A comparison of two chronosequences of rehabilitated coal discard sites was achieved by an application of the 'space-for-time' hypothesis. Sites of different ages and at separate locations ('space') were identified to obtain a chronosequence of ages ('time'). The two chronosequences included sites aged 1 to 11 years (chronosequence A) and 6 to 17 years (chronosequence B), respectively. Sites in the same chronosequence were managed identically, while there was a distinct difference in management practices applied to each chronosequence. The long-term effect of the different management regimes on the soil microbial community function and structure was investigated. Again, there was no relationship between rehabilitation age and microbial community measurements. Fluctuations of selected microbial properties occurred in both chronosequences and similar temporal trends existed over the rehabilitation periods. However, the less intensively managed chronosequence (8) seemed more stable (less fluctuation occurred) over the rehabilitation period than the more intensively managed chronosequence (A). It was therefore concluded that the microbial communities in the less managed sites maintained their functional and structural integrity within bounds in the absence of management inputs or disturbance. While there was similarity in the trends over time for individual microbial community measurements, the seemingly more stable conditions in chronosequence 6 are important in terms of the goal of rehabilitation. / Thesis (Ph.D. (Environmental Science)--North-West University, Potchefstroom Campus, 2007

Biofuel cells (BFCs) that utilize enzymes as catalysts represent a new sustainable and renewable energy technology. Numerous efforts have been directed to improve the performance of the enzymatic BFCs (EBFCs) with respect to power output and operational stability for further applications in portable power sources, self-powered electrochemical sensing, implantable medical devices, etc. This concept article details the latest advances about the EBFCs based on porous nanoarchitectures over the past 5 years. Porous matrices from carbon, noble metal, and polymer promote the development of EBFCs through the electron transfer and mass transport benefits. We will also discuss some key issues on how these nanostructured porous media improve the performance of EBFCs in the end.

The work of the present thesis focused on conversion of the cellulosic part of waste textiles into biogas and ethanol, and its challenges. In 2009, the global annual fiber consumption exceeded 70 Mt, of which around 40% consisted of cellulosic material. This huge amount of fibers is processed into apparel, home textiles, and industrial products, ending up as waste after a certain time delay. Regretfully, current management of waste textiles mainly comprises incineration and landfilling, in spite of the potential of cellulosic material being used in the production of ethanol or methane. The volume of cellulose mentioned above would be sufficient for producing around 20 billion liters of ethanol or 11.6 billion Nm3 of methane per year. Nevertheless, waste textiles are not yet accepted as a suitable substrate for biofuel production, since their processing to biofuel presents certain challenges, e.g. high crystallinity of cotton cellulose, presence of dyes, reagents and other materials, and being textiles as a mixture of natural and synthetic fibers. High crystallinity of cotton cellulose curbs high efficient conversion by enzymatic or bacterial hydrolysis, and the presence of non-cellulosic fibers may create several processing problems. The work of the present thesis centered on these challenges. Cotton linter and blue jeans waste textiles, all practically pure cellulose, were converted to ethanol by SSSF, using S. cerevisiae, with a yield of about 0.14 g ethanol/g textile, only 25% of the theoretical yield. To improve the yield, a pretreatment process was required and thus, several methods were examined. Alkaline pretreatments significantly improved the yield of hydrolysis and subsequent ethanol production, the most effective condition being treatment with a 12% NaOH-solution at 0 °C, increasing the yield to 0.48 g ethanol/g textile (85% of the theoretical yield). Waste textile streams, however, are mixtures of different fibers, and a separation of the cellulosic fibers from synthetic fibers is thus necessary. The separation was not achieved using an alkaline pretreatment, and hence another approach was investigated, viz. pretreatment with N-methyl-morpholine-N-oxide (NMMO), an industrially available and environment friendly cellulose solvent. The dissolution process was performed under different conditions in terms of solvent concentration, temperature, and duration. Pretreatment with 85% NMMO at 120 °C under atmospheric pressure for 2.5 hours, improved the ethanol yield by 150%, compared to the yield of untreated cellulose. This pretreatment proved to be of major advantage, as it provided a method for dissolving and then recovering the cellulose. Using this method as a foundation, a novel process was developed, refined and verified, by testing polyester/cellulose-blended textiles, which predominate waste textiles. The polyesters were purified as fibers after the NMMO treatments, and up to 95% of the cellulose content was regenerated. The solvent was then recovered, recycled, and reused. Furthermore, investigating the effect of this treatment on anaerobic digestion of cellulose disclosed a remarkable enhancement of the microbial solubilization; the rate in pretreated textiles was twice the rate in untreated material. The overall yield of methane was, however, not significantly affected. The process developed in the present thesis appears promising for transformation of waste textiles into a suitable raw material, to subsequently be used for biological conversion to ethanol and biogas. / <p>Thesis to be defended in public on Friday, May 27, 2011 at 13.00 at KC-salen, Kemigården 4, Göteborg, for the degree of Doctor of Philosophy.</p>

The human patogen Streptococcus pneumoniae encodes a single copy of eukaryotic-like Ser/Thr protein kinase StkP. StkP regulates virulence, competence, stress resistence, gene expression and plays a role in the regulation of cell division cycle. Analysis of phosphoproteome maps of the wild type and stkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including Mn-dependent inorganic pyrophosphatase PpaC. Mass spectrometry analysis identified two phosphorylation sites in an active site of the protein. Pyrophosphatases are essential enzymes that catalyze hydrolysis of inorganic pyrophosphate produced during various biosynthetic reactions that utilize ATP. Changes in pyrophosphatase activity have been described to have global effects on cell metabolism, growth and division of bacteria. The aim of this thesis was to investigate the phosphorylation of inorganic pyrophosphatase PpaC in S. pneumoniae. Gene ppaC was cloned, expressed in E. coli and protein was purified via affinity chromatography. Phosphorylation of PpaC by StkP was examined in a kinase assay but we did not confirm that PpaC is a direct substrate of StkP in vitro. Further we prepared a set of mutants in ppaC gene. We replaced two presumable phosphoaminoacids identified by mass-spectrometry with...

Ce travail de thèse s'inscrit dans le cadre de la valorisation des co-produits issus des industries de transformation et de conserve de thon. Il porte sur la mise en oeuvre de l'hydrolyse enzymatique et des procédés de séparation membranaires en vue d'obtenir des composés d'intérêt comme les peptides et les acides aminés. Les techniques utilisées dans ce travail font partie des « technologie propre », visant une dépense énergétique et un investissement modérés. L'hydrolyse enzymatique a été menée pour identifier l'efficacité des enzymes sur la matrice afin de déterminer les conditions optimales d'hydrolyse en utilisant la méthodologie des plans d'expériences avec pour objectif l'enrichissement de la phase soluble en peptides de petite taille dotés d'activités biologiques intéressantes. Le fractionnement par la taille par ultrafiltration et Nanofiltration a été utilisé comme un procédé pour agir sur la distribution de la taille moléculaire de la population peptidique et sur les propriétés d'hydrolysat produit. Tout d'abord, un fractionnement à petite échelle a été réalisé avec des membranes de différents seuils de coupure et de différentes natures (organique et inorganique) afin de sélectionner la meilleure membrane répondant à notre objectif.Ensuite une étude d'optimisation des conditions opératoire a été réalisée pour les deux étapes de fractionnement UF et NF de manière à déterminer les meilleures conditions séparatives en utilisant un hydrolysat commercial. La validation du procédé de fractionnement en utilisant l'hydrolysat des co-produits de thon a été effectuée. Durant cette étude, différents modes de fractionnement utilisant différentes combinaisons d'UF et NFont été testés pour déterminer le meilleur procédé permettant la récupération du maximum de fractions peptidiques intéressantes. L'originalité de ce travail de thèse réside d'une part, dans l'enrichissement de l'hydrolysat des co-produit de thon en composés valorisables comme les acides aminés essentiels ainsi que les peptides dotés de valeur ajoutée et d'autre part, la mise en place d'un procédé de fractionnement pour la récupération des différentes fractions afin de mettre en évidence la conception d'un bioréacteurs enzymatique couplé à la technique membranaire. / This work is performed in the framework of up-grading of tuna by-products generated from processing and conditioning industries. The enzymatic hydrolysis combined with membrane separation processes in order to obtain the fraction of interest peptides and amino acids was studied. The optimal conditions during enzymatic hydrolysis were determined using the methodology of design of experiments in order to enrich the soluble phase in small peptides with interesting biological activities. The fractionation by Ultrafiltration and Nanofiltration following a suitable combination was studied. For this, firstly, a small-scale fractionation was performed with membranes of different cut-off and different natures (organic and inorganic) to select the best membrane processes combination and to optimize the used conditions. Then, a validation study of the fractionation using the hydrolysate of tuna by-products produced during was performed. In this study, different modes of fractionation combination of concentration and diafiltration steps were tested to determine the best method for the recovery of large quantities of interesting peptide fractions. The originality of this PhD work is the enrichment of the tuna by-products hydrolysate with valuable compounds such as essential amino acids and peptides with a high biological activity.

This thesis has been focused on enzymatic kinetic resolutions and stereoselective oxidative transformations of enallenes catalyzed by PdII. In the first part of the thesis, a detailed discussion on Candida antarctica lipase B (CALB)-catalyzed kinetic resolution (KR) of δ-functionalized alkan-2-acetates is shown. We gained a deeper insight into the mechanism of enzyme-substrate recognition. Changing from an anhydrous solvent to water or a water-containing organic solvent enhanced the enantioselectivity. The effect of –OH was also confirmed by a lipase mutant suggesting that the water molecule mentioned above can be partly mimicked. In the second part of the thesis, we developed an efficient KR for allenic alcohols. On this basis, a novel synthesis of optically pure 2-substituted 2,3-dihydrofurans from allenic alcohols via a Ru-catalyzed cycloisomerization was reported. The developed protocol enabled us to assemble an optically pure precursor for total synthesis with three chiral centers from readily available allenol in 2 days. In the third part, we reported a class of reactions involving C–H cleavage under mild conditions: PdII-catalyzed oxidative transformations of enallenes. These reactions are particularly attractive since a number of meticulous structures have been achieved from readily accessible starting materials. The directing effect of an unsaturated hydrocarbon was found to be key for these transformations. In the final part, we developed the carbonylative insertion reaction discussed in the third part of the thesis into an asymmetric version. By using this methodology, a number of cyclopentenone compounds were obtained in good to excellent enantioselectivity.

Doctor of Philosophy / Department of Biological & Agricultural Engineering / Donghai Wang / Lignocellulosic biomass such as agricultural residues tends to be a sustainable feedstock for production of biofuels and biobased products in the long term due to its high availability and relative low cost. However, conversion of lignocellulosic biomass to biofuels faces significant technical challenges. One of the major challenges is biomass logistics. The agricultural residues are often harvested during a limited harvest season and stored as bales with low bulk density, making them difficult to handle, transport, store, and use in their natural forms. Densification of biomass by pelleting process could significantly improve the bulk density of biomass and thus improve handling efficiency and reduce transportation and handing costs. The main focus of this research was to better understand the impacts of pelleting process as well as pelleting conditions on physical properties, chemical compositions, biomass structure, and fermentable sugar yield of sorghum stalk, corn stover, wheat straw, and big bluestem. Results showed that pelleting process can increase biomass density up to 9-12 folds. Pelleting condition such as hammer mill screen size and ring-die pelleting mill die thickness had significant effects on bulk density, true density, and durability of biomass pellets. Although the pelleting process did not show significant effects on chemical composition of biomass before dilute-acid pretreatment process, glucan content of biomass pellets increased with the increase in ring-die pelleting mill die thickness and decreased with the increase in mill screen size after dilute-acid pretreatment. Opposite trend was observed for xylan content of biomass pellets as affected by pelleting conditions after dilute-acid pretreatment process. Biomass crystallinity increased after pelleting process, but not in a significant level. Softened surface region of biomass was removed after pelleting process, making the biomass more amendable to enzymatic attack. In this study, the optimum pelleting conditions were to grind the biomass feed using a 6.5-mm mill screen and to pellet biomass using a 44.5-mm ring-die pelleting mill die thickness. Under this optimum pelleting condition, the enzymatic conversion of cellulose of wheat straw pellets was the highest (94.1%), followed by corn stover pellet (93.1%), sorghum stalk pellet (92.1%), and big bluestem pellet (91.1%).