Flavonoid glycosyltransferases (GTs), enzymes integral to plant ecological responses and human pharmacology, necessitate rigorous structural elucidation to decipher their mechanistic function and substrate specificity, particularly given their role in the biotransformation of diverse pharmacological agents and natural products. This investigation delved into a comprehensive exploration of the flavonol 3-O GT from Citrus paradisi (Cp3GT), scrutinizing the impact of a c-terminal c-myc/6x histidine tag on its enzymatic activity and substrate specificity, and successfully achieving its purification to apparent homogeneity. This established a strong foundation for potential future crystallographic and other structure/function analyses. Through the strategic implementation of site-directed mutagenesis, a thrombin cleavage site was incorporated proximal to the tag, followed by cloning in Pichia pastoris, methanol-induced expression, and cobalt-affinity chromatography for initial purification stages. Notably, the recombinant tags did not exhibit a discernible influence on Cp3GT kinetics, substrate preference, pH optima, or metal interactions, maintaining its specificity towards flavonols at the 3-OH position and favoring glucosylation of quercetin and kaempferol. Subsequent purification steps, including MonoQ anion exchange and size-exclusion chromatography, yielded Cp3GT with ≥95% homogeneity. In silico molecular models of Cp3GT and its truncated variants, Cp3GTΔ80 and Cp3GTΔ10, were constructed using D-I-TASSER and COFACTOR to assess binding interactions with quercetin and kaempferol. Results indicated minimal interference of c-myc/6x-his tags with the native Cp3GT structure. This study not only lays a foundation for impending crystallographic studies, aiming to solidify the understanding of Cp3GT's stringent 3-O flavonol specificity, but also accentuates the potential of microbial expression platforms and plant metabolic engineering in producing beneficial compounds. To this end, a thorough review of four pivotal classes of plant secondary metabolites, flavonoids, alkaloids, betalains, and glucosinolates, was conducted. This will open avenues for further research and applications in biotechnological, medical, and agricultural domains.

Tunicamycin nucleoside antibiotics were the first known to target the formation of peptidoglycan precursor lipid I in bacterial cell wall biosynthesis. They have also been used extensively as inhibitors of protein N-glycosylation in eukaryotes, blocking the biogenesis of early intermediate dolichyl-pyrophosphoryl-N-acetylglucosamine. Despite their unusual structures and useful biological properties, little is known about their biosynthesis. Elucidating the metabolic pathway of tunicamycins and gaining an understanding of the enzymes involved in key bond forming processes would not only be of great academic value in itself, it would also unlock a comprehensive toolbox of biosynthetic machinery for the production of tunicamycin analogues which have the potential to act as novel therapeutic antibiotics or as specific inhibitors of medicinally important NDP-dependent glycosyltransferases. I – Cloning the tunicamycin biosynthetic gene cluster. We report identification of the tunicamycin biosynthetic genes in Streptomyces chartreusis following genome sequencing and a chemically-guided strategy for in silico genome mining that allowed rapid identification and unification of an operon fractured across contigs. Heterologous expression established a likely minimal gene set necessary for antibiotic production, from which a detailed metabolic pathway for tunicamycin biosynthesis is proposed. II – Natural product isolation and degradation. We have developed efficient methods for the isolation of tunicamycins from liquid culture in preparative quantities. A subsequent relay synthesis furnished advanced biosynthetic intermediates for use as precursors in the production of tunicamycin analogues and as substrates for the in vitro characterisation of individual Tun enzymes. III – Functional characterisation of tun gene products. Individual tun gene products were over-expressed and purified from recombinant E. coli hosts, allowing in vitro functional studies to take place. An NMR assay of biosynthetic enzyme TunF showed it acted as a UDP-GlcNAc-4-epimerase. Putative glycosyltransferase TunD showed hydrolytic activity towards substrate UDP-GlcNAc but failed to accept to the expected natural acceptor substrate, providing unexpected insights into the ordering of biosynthetic events in the tunicamycin pathway. Initial studies into the over-expression of the putative sugar N-deacetylase TunE were also described. IV – Towards synthesis of tunicamycin fragments. Investigations into a novel synthesis of D-galactosamine – a structural motif within tunicamycin – led to the unexpected observation of inverted regioselectivity upon RhII-catalysed C-H insertion of a D-mannose-derived sulfamate. This was the first example of N-insertion at the beta- rather than gamma-C-H based on conformation alone and warranted further investigation. The X-ray structure of a key sulfamate precursor offered valuable insights as to the source of this unique selectivity.

A ciclodextrina glicosiltransferase [E.C. 2.4.1.19; CGTase] é uma enzima industrialmente importante, usada para produzir ciclodextrinas. Neste trabalho foi utilizado o planejamento experimental e a metodologia de superfície de resposta a fim de identificar as melhores condições para a produção de CGTase pela bactéria alcalófila Bacillus circulans. As melhores condições de temperatura e pH, para a produção de CGTase foram, respectivamente, 36 ºC e 9,7. Em seguida, utilizando estes parâmetros otimizados, a produção de CGTase foi avaliada em cultivos submersos, batelada e batelada alimentada, e em cultivo semi-sólido (CSS), usando um resíduo fibroso de soja (SIFR) como substrato. Nos cultivos em batelada, a atividade máxima de CGTase obtida foi de 1155 U.mL-1, em aerobiose. A produção de CGTase foi bastante influenciada pelo fluxo de ar e pela agitação, sendo que uma alta produtividade enzimática (155 U.(mLh)-1) foi obtida em condições de aeração moderada (400 rpm para a velocidade de agitação e 1,7 vvm para o fluxo de ar). Com estes parâmetros otimizados, a produtividade de CGTase obtida em cultivo em batelada alimentada foi de 137 U.(mLh)-1, com uma taxa de alimentação de 0,17 g.(Lh)-1. O crescimento celular e a síntese de CGTase, usando o resíduo fibroso de soja como substrato apresentou um rendimento de 32.776 U.g(SIFR) -1. As diferentes abordagens utilizadas neste trabalho poderão ser aplicadas para a produção de outras enzimas amilolíticas e também para a produção de CGTase com outros substratos. / Cyclodextrin glycosyltransferase [E.C. 2.4.1.19; CGTase] is an industrially important enzyme, which is used to produce cyclodextrins. In this study we report the use of experimental factorial design and response surface methodology to find the best conditions for CGTase production by alkaliphilic Bacillus circulans. The optimized calculated values for the tested variables were pH 9.7 and temperature 36oC. The CGTase production was further studied with the optimized process parameters on submerged cultivations (SC), batch or fedbatch, and solid-state cultivations (SSC) using soybean industrial fibrous residue (SIFR). The maximum CGTase activity obtained on batch cultivation was 1,155 U.mL-1 under aerobic conditions. The CGTase production was strongly affected by air flow rate and agitation speed, showing high enzyme productivity (155 U.mL-1h-1) under moderate conditions of aeration (400 rpm for speed agitation and 1.7 vvm for air flow rate). With these optimized process parameters, CGTase productivity obtained on fed-batch cultivations was 137 U.mL-1h-1, with feeding rate at 0.17 g.L-1h-1. Cell growth and CGTase synthesis in SSC using soybean industrial fibrous residue as substrate was excellent, with CGTase yield of 32,776 U.g(SIFR) -1. The different approaches used in this study may also find applications for the production of other starch-converting enzymes and also for other CGTase-producing substrates.

Ce travail cherche à apporter un éclairage sur les relations séquence-structure-fonction des alpha1,3/4-fucosyltransférases, avec un accent particulier sur les core alpha1,3-fucosyltransférases des plantes. La fucosylation de type Core alpha1,3 est une caractéristique des oligosaccharides N-liés des plantes et invertébrés, avec une fonction biologique qui n'est pas encore élucidée. L'activité Core alpha1,3-fucosyltransférase est responsable d'allergies alimentaires, au pollen, et aux insectes chez l'homme. Dans le cadre de ce travail sont présentés des résultats de caractérisation biochimique (effet de cations divalents sur l'activité, Km de substrat donneur), des expériences de troncation des différents domaines (ex: suppression du domaine C-terminal spécifique aux core alpha1,3-fucosyltransférases des plantes), et de mutagenèse dirigée, en utilisant comme protéine modèle, la core 1,3-fucosyltransferase A (FucTA) d'Arabidopsis thaliana qui a été exprimée sous forme recombinante chez Pichia pastoris. Ces expériences ont été dictées sur la base de nos résultats d'analyses bioinformatiques des séquences de alpha1,3/4-fucosyltransférases et de la modélisation par homologie du domaine de liaison au nucléotide-sucre de l'enzyme FucTA. La mutagenèse des résidus clé identifiés par cette approche a permis de confirmer l'importance de certains acides aminés dans le mécanisme catalytique. Enfin la protéine FucTA étant elle-même glycosylée quand elle est produite chez P. pastoris, nous avons étudié l'impact de cette glycosylation sur la production et l'activité de la protéine, par des expériences de mutagenèse, de Western blotting et de spectrométrie de masse.

Wood formation or xylogenesis is a fundamental process for so diverse issues as industry, shelter and a sustainable environment. Wood is comprised of secondary xylem, rigid large cells with thick cell walls that are lignified. The basis for the sturdy cells is an advanced composite made up of cellulose fibers cross-linked by hemicelluloses and finally embedded in lignin. This fiber-composite is the secondary cell walls of woody plants. Cell division and differentiation is regulated by switching on and off genes. Proteins encoded by these genes execute the major functions in the cells. They steer the entire machinery operating the structure and function of the cells, maintaining growth and synthesising essential products such as the cell wall carbohydrates.   Here we describe the investigation of genes and proteins involved in xylan formation as well as the development of a model system that will aid the functional analysis of wood formation. Xylan is the main hemicellulose or cross linking glycan in dicot wood and thereby one of the most abundant carbohydrates on earth. We demonstrate that hybrid aspen cell suspension cultures can be used as a model system for secondary cell wall formation. We have also examined glycosyltransferases from CAZy family 43 that play a part in secondary cell wall formation. We have focused on one of these, Pt×tGT43A, a likely ortholog of Arabidopsis IRX9, which plays a crucial role in xylan formation. The protein was transiently expressed in Nicotiana benthamiana and its function and localization is described. Also, we investigate a glycoside hydrolase, Pt×tXyn10A, involved in wood formation. Its role is not clear but it most likely modifies xylan as it gets incorporated into the secondary cell wall after secretion from the Golgi. This influences the interaction between cellulose, xylan and lignin in the finished wood cell. We have also cloned a transcription factor, Pt×tMYB021, a likely ortholog of Arabidopsis MYB46 and we show that it activates GT43A, GT43B and Xyn10A. By analysis of the promoter sequences we identify a CA-rich motif putatively important for xylem-specific genes.   By mastering proteins involved in xylogenesis we will acquire the tools to improve and develop the wood product market. Xylan is an immense unexploited source of renewable carbohydrate. New products envisioned include e.g. faster growing trees, changed fiber characteristics, optimised utilization of wood carbohydrates for biofuels and biomaterials as well as invention of intelligent materials by biomimetic engineering. / Vedbildning, eller xylogenes, är en grundläggande mekanism för så skilda områden som industri, boende och en hållbar miljö. Ved består av sekundärt xylem som är starka, stora celler med tjocka cellväggar som är lignifierade. Grunden för de starka cellerna är en avancerad komposit bestående av cellulosafibrer tvärbundna av hemicellulosa och slutligen ingjutet i lignin. Denna fiberkomposit är den sekundära cellväggen i vedartade växter. Celldelning och differentiering regleras genom att sätta igång och stänga av gener. Proteiner som kodas av dessa gener utför de viktigaste funktionerna i cellerna. De styr hela maskineriet som upprätthåller cellernas struktur och funktion, underhåller tillväxt samt tillverkar nödvändiga produkter såsom cellväggskolhydraterna. Här beskriver vi utforskningen av gener och proteiner som är inblandade i xylanbildning liksom utvecklandet av ett modellsystem som kommer vara en hjälp i den funktionella analysen av vedbildning. Xylan är den vanligaste hemicellulosan, eller korsbindande glykanen, i lövträd och därför en av de vanligaste kolhydraterna på jorden. Vi demonstrerar att hybridaspcellkulturer i suspension kan användas som ett modellsystem för sekundär cellväggsbildning. Vi har också undersökt glykosyltransferaser från CAZy-familj 43 som tycks spela en viktig roll i bildandet av sekundär cellvägg. Vi har fokuserat på en av dessa, Pt×tGT43A, en trolig ortolog till Arabidopsis IRX9 som spelar en viktig roll i xylanbildning. Proteinet har uttryckts övergående i Nicotiana benthamiana och dess funktion och lokalisering beskrivs. Dessutom undersöker vi ett glykosidhydrolas, Pt×tXyn10A, involverad i vedbildning. Dess roll är oklar men högst sannolikt modifierar det xylan medan det inkorporeras i sekundära cellväggen efter sekretion från Golgi. Detta influerar interaktionen mellan cellulosa, hemicellulosa och lignin i den slutliga vedcellen. Vi har också klonat en transkriptionsfaktor, Pt×tMYB021, en trolig ortolog till Arabidopsis MYB46 och vi visar att den aktiverar GT43A, GT43B och Xyn10A. Genom analys av promotorsekvenserna har vi identifierat ett CA-rikt motiv förmodat viktigt för xylemspecifika gener.Genom att bemästra proteinerna som är ansvariga för vedbildning får vi verktyg att utveckla skogsproduktsmarknaden. Xylan är en ofantligt stor outnyttjad källa till förnyelsebara kolhydrater. En vision är nya produkter som till exempel snabbväxande träd, ändrade fiberegenskaper, optimerat användande av vedkolhydrater för biobränsle och biomaterial såväl som utvecklandet av intelligenta material genom biomimetisk ingenjörskonst. / QC20100730

Aquesta tesi se centra en l’estudi de les possibles glicosiltransferases de Micoplasma genitalium involucrades en la síntesi de glicolípids. Aquests compostos formen part de la membrana plasmàtica del microorganisme, únic envolcall que el protegeix del seu entorn ja que no disposa de paret cel•lular. La hipòtesi sobre la qual s’ha desenvolupat el treball és la possibilitat que aquests glicolípids i els enzims encarregats de la seva producció, les glicosiltransferases, siguin essencials per a la viabilitat del micoplasma i per tant, la seva inhibició sigui una forma d’eradicar les infeccions causades pel patògen. De les tres seqüències classificades com a glicosiltrasferases en el genoma de Mycoplasma genitalium, mg025, mg060 i mg517, s’ha determinat que mg025 és l’únic dels tres gens que no és essencial per al bacteri, tot i que s’ha observat que els tres s’expressen tant a la fase exponencial com a la fase estacionària del seu creixement. La funció de mg025 i mg060 és encara desconeguda, mentre que mg517 és la glicosiltransferasa encarregada de la síntesi dels dos principals glicolípids del micoplasma, el monoglicosildiacilglicerol (MGlcDG) i el diglicosildiacilglicerol (DGlcDG). En aquesta tesi s’ha desenvolupat un protocol d’expressió per a la glicosiltransferasa codificada per mg517, anomenada GT-MG517, el qual fa ús d’una coexpressió amb xaperones i solubilitza la proteïna amb detergents, glicerol i una elevada força iònica. Aquesta metodologia ha estat necessària ja que GT-MG517 és una proteïna associada a membrana i la seva expressió recombinant en E.coli presenta dificultats. La proteïna s’ha purificat mitjançant cromatografia d’afinitat per Ni, tot i que el grau de puresa assolit no ha estat suficient per a intentar la seva cristal•lització. Les diverses proves realitzades usant cromatografia d’exclusió molecular han permès determinar que GT-MG517 forma oligòmers d’alt pes molecular. A partir de la proteïna purificada s’ha realitzat un estudi cinètic de la seva doble activitat glicosildiacilglicerolsintasa. D’aquest estudi s’extreu que GT-MG517 pot transferir un sucre, Glc o Gal, a una molècula principalment hidrofòbica, com és el DOG, i a una molècula molt més hidrofílica, com el MGlcDG. Ambdós compostos però posseeixen un alcohol primari sobre el qual té lloc la transferència creant l’enllaç (16). Amb qualsevol dels substrats acceptors provats, l’enzim presenta activitats específiques superiors si el substrat donador és UDP-Gal. L’acceptor preferit de GT-MG517 és el lípid DOG, però la glicosiltransferasa és capaç d’elongar, ja sigui amb una Glc o amb una Gal, glicolípids com el MGlcDG i el MGDEG, preferint en aquest cas el compost amb una Gal a l’extrem no reductor. Tot i això, l’afinitat de l’enzim és superior per a donadors amb Glc (KM inferiors a les dels donadors amb Gal). D’altra banda, s’ha demostrat que el lípid aniònic DOPG es comporta com a activador de l’activitat enzimática. GT-MG517 posseeix un teòric domini d’unió d’UDP-Glc a l’extrem N-terminal. Aquesta zona presenta similitud de seqüència amb altres glicosiltransferases i permet la classificació de GT-MG517 dins de la família GT-2. En canvi, el seu extrem C-terminal presenta una seqüència particular que no s’alinea amb altres proteïnes. En aquesta tesi es formula la hipòtesi que aquesta zona podria ser d’interacció amb la membrana i, a més, que aquesta interacció podria regular l’activitat enzimàtica. Per això es preparen diverses formes truncades de GT-MG517, en les quals s’eliminen alguns aminoàcids de l’extrem C-terminal, i una forma en la qual només es conserva l’hipotètic domini d’unió d’UDP-Glc. Aquestes noves proteïnes s’expressen, solubilitzen i purifiquen aplicant el protocol establert per a la forma completa. Amb l’anàlisi dels glicolípids sintetitzats s’estableix que els deu últims aminoàcids de GT-MG517 són prescindibles per a la seva activitat, ja que les formes truncades corresponents continuen tenint la capacitat de sintetitzar glicolípids. No obstant, l’eliminació d’un nombre superior de residus, inactiva la proteïna. La purificació de l’hipotètic domini d’unió d’UDP-Glc posa de manifest que forma oligòmers, de la mateixa forma que ho fa la proteïna completa. / Esta tesis se centra en el estudio de las posibles glicosiltransferasas de Mycoplasma genitalium involucradas en la síntesis de glicolípidos. Estas moléculas constituyen parte de la membrana plasmática del miroorganismo, única estructura de protección frente al entorno ya que los micoplasmas carecen de pared celular. La hipótesis sobre la cual se ha desarrollado el trabajo es la posibilidad que los mencionados glicolípidos y las enzimas encargadas de su producción, las glicosiltransferasas, sean esenciales para la viabilidad del micoplasma y, por tanto, su inhibición sea una forma de erradicar las infecciones causadas por el patógeno. De las tres secuencias clasificadas como glicosiltransferasas en el genoma de Mycoplasma genitalium, mg025, mg060 y mg517, se determinó que mg025 es el único de los tres genes que no es esencial para la bacteria, aunque se observó que los tres se expresan tanto en la fase exponencial como en la fase estacionaria de su crecimiento. Se desconoce todavía la función de mg025 y mg060. En cambio, se conoce que mg517 es la glicosiltransferasa responsable de la síntesis de los dos principales glicolípidos del micoplasma, el monoglicosildiacilglicerol (MGlcDG) y el diglicosildiacilglicerol (DGlcDG). En esta tesis se desarrolló un protocolo de expresión para la glicosiltransferasa codificada por mg517, llamada GT-MG517, el cual emplea una coexpresión con chaperonas y solubiliza la proteína con detergentes, glicerol y una fuerza iónica elevada. Dicha metodología fue necesaria ya que GT-MG517 es una proteína asociada a membrana y su expresión recombinante en E.coli presenta dificultades. La proteína se purificó mediante cromatografía de afinidad por Ni pero el grado de pureza obtenido no fue suficiente para intentar su cristalización. Distintas pruebas realizadas usando cromatografía d’exclusión molecular permitieron determinar que GT-MG517 forma oligómeros de alto peso molecular. Con la proteína purificada se realizó un estudio cinético de su doble actividad glicosildiacilglicerolsintasa. De este estudio se extrae que GT-MG517 puede transferir un azúcar, Glc o Gal, a una molécula principalmente hidrofóbica, como es el DOG, y a una molécula mucho más hidrofílica, como el MGlcDG. Pese a su diferencia, los dos compuestos poseen un alcohol primario sobre el que tiene lugar la transferencia creándose el enlace (16). Con cualquiera de los sustratos aceptores probados, la enzima presenta actividades específicas superiores si el sustrato dador es UDP-Gal. El aceptor preferido de GT-MG517 es el lípido DOG, aunque la glicosiltransferasa es capaz de elongar, ya sea con una Glc o con una Gal, glicolípidos como el MGlcDG y el MGDEG, prefiriendo en este caso el compuesto con una Gal en el extremo no reductor. Aún así, la afinidad de la enzima es superior para dadores con Glc (KM inferiores a las de los dadores con Gal). Por otro lado, se demostró que el lípido aniónico DOPG se comporta como activador de la actividad enzimática. GT-MG517 posee un dominio teórico de unión de UDP-Glc en su extremo N-terminal. Esta zona presenta similitud de secuencia con otras glicosiltransferasas y permite la clasificación de GT-MG517 dentro de la familia GT-2. En cambio, su extremo C-terminal presenta una secuencia particular que no se alinea con otras proteínas. En esta tesis se formula la hipótesis que dicha zona podría ser de interacción con la membrana y, además, que dicha interacción podría regular la actividad enzimática. Por eso se prepararon distintas formas trucadas de GT-MG517, en las cuales se eliminaron algunos aminoácidos del extremo C-terminal, y una forma en la cual sólo se conservó el hipotético dominio de unión de UDP-Glc. Las nuevas proteínas se expresaron, solubilizaron y purificaron aplicando el protocolo establecido para la forma completa. Con el análisis de los glicolípidos sintetizados se determinó que los diez últimos aminoácidos de GT-MG517 eran prescindibles para su actividad, ya que las correspondientes formas truncadas conservaban su capacidad de sintetizar glicolípidos. No obstante, la eliminación de un número superior de residuos inactiva la proteína. La purificación del hipotético dominio de unión de UDP puso de manifiesto que forma oligómeros, de la misma forma en que lo hace la proteína completa. / This thesis is focused on the glycolipid producing glycosyltransferases from Mycoplasma genitalium. Glycolipids are part of the microorganism plasma membrane, which is the external covering of mycoplasmas since they lack a cell wall. Our working hypothesis is that glycolipids and the enzymes responsible for their production are essential to mycoplasma. Thus, glycosyltransferase inhibition could be a way to eradicate this pathogen caused infections. There are three genes described as putative glycosyltransferases in Mycoplasma genitalium genome, mg025, mg060 and mg517. We determined that mg025 is the only one essential to the bacteria, although the three of them are expressed both during the exponential and stationary growth phases. The function of mg025 and mg060 still remains unknown, whereas mg517 codifies for the glycosyltransferase responsible for monoglycosyldiacylglycerol (MGlcDG) and diglycosyldiacylglycerol (DGlcDG) synthesis, main mycoplasma glycolipids. In this work an expression protocol for mg517 codified glycosyltransferase was designed. The protocol included chaperone coexpression and protein solubilization with detergents, glycerol and high ionic strength. This methodology was necessary since GT-MG517 is a membrane associated protein and its recombinant expression in E.coli presents some difficulties. GT-MG517 was purified by Ni affinity chromatography. However, a suitable purity degree to attempt protein crystallization was not achieved. Some experiments using size exclusion chromatography revealed that GT-MG517 forms high molecular weight oligomers. A kinetic study of the protein double glycosyldiacylglycerol synthase activity was performed. From this study we learned that GT-MG517 is able to transfer a sugar moiety, Glc or Gal, both to a hydrophobic molecule such as DOG or to a more hydrophilic compound such as MGlcDG. These acceptor substrates share a primary alcohol where the sugar transfer takes place forming a (16) bond. The enzyme presents higher specific activities when UDP-Gal acts as reaction donor, regardless of the acceptor tested. DOG is the preferred acceptor although the enzyme is able to transfer Glc or Gal moieties to glycolipids such as MGlcDG and MGDEG. In this case, the reaction is faster with acceptors with Gal in the non-reducing end. As for donor substrate, enzyme’s affinity is higher for Glc containing molecules, with lower KM values than for Gal donors. In addition, our study proved the anionic lipid DOPG to act as an enzymatic activity enhancer. GT-MG517 has a putative binding domain for UDP-Glc in the N-terminal end. This sequence is similar to other glycosyltransferases and allows its classification in Cazy’s GT-2 family. On the contrary, the C-terminal end has a particular sequence which does not match up with any other protein. Our hypothesis was that this C-terminal end of GT-MG517 could contain a membrane interaction sequence, which could at the same time modulate enzymatic activity. To test this hypothesis some truncated forms of GT-MG517, where C-terminal aminoacids had been removed, were prepared. Moreover, a form where only the putative UDP-Glc binding domain was conserved was also expressed. All these proteins were expressed, solubilized and purified with the same protocol used for the full-length form of GT-MG517. Glycolipids produced by truncated forms were analysed and results implied that the last ten aminoacids were not involved in the enzyme’s activity. Elimination of a higher number of aminoacids caused protein inactivation. When the putative UDP-binding domain was purified, it showed high molecular weight oligomers such as those developed by the complete form of the protein.

The Arabidopsis thaliana glycosyltransferase UGT72B1 is one of many enzymes which catalyze the reaction oflinking a glucose moiety from UDP-glucose to an acceptor molecule, in this case a chloroaniline or a chlorophenol. This is part of a detoxification system of the plant cell, similar to that in humans where a glucuronosyltransferases are enabling drug metabolism. It would be of interest to investigate the activity of the human enzyme towards different pharmaceuticals and determine the effect the linkage of glucose has to properties of the compounds. However, the human enzymes are membrane proteins and thus difficult to purify and crystallize. Here, an attempt was made to instead change the substrate specificity of UGT72B1 from UDPglucose to UDP-glucuronic acid. Combination of the four point mutations G18S, P139R, W367S and AG387ED were introduced in UGT72B1. However, no UDP-glucuronic acid activity was obtained. Single mutants W367S and AG387ED retained similar activity as of the wildtype while P139R had highly reduced activity and G18S was not expressed at all. All other combinations of mutations resulted in even less activity. Four chimeric proteins were also constructed. They were combinations of the UGT72B1 and the human enzyme UGT2B4. These were all soluble proteins but no activity could be determined. / Glykosyltransferaset UGT72B1 från Arabidopsis thaliana är ett av många enzymer som katalyserar reaktionen där en glukosenhet från UDP-glukos länkas till en acceptormolekyl, i det här fallet en kloranilin eller en klorfenol. Det är en del av ett detoxifieringssytem i växtcellen, som liknar det i människan, där ett glukuronosyltransferas möjliggör nedbrytning av bl.a. läkemedel. Det vore intressant att kunna undersöka de humana enzymernas aktivitet mot olika läkemedel och även fastställa effekten glukoslänkningen har på dessa substansers egenskaper. De humana enzymerna är dock membranprotein och är därför svåra att rena fram och att kristallisera. Här har istället ett försök gjorts för att ändra substratspecificiteten hos UGT72B1 från UDP-glukos till UDP-glukuronsyra. Kombinationer av de fyra punktmutationerna G18S, P139R, W367S och AG387ED introducerades i UGT72B1. Ingen aktivitet med UDP-glukuronsyra erhölls dock. Enkelmutanterna W367S och AG387ED bibehöll liknande aktivitet som vildtypen, medan P139R hade starkt reducerad aktivitet och G18S uttrycktes inte alls. Alla andra kombinationer av mutationer resulterade i ännu lägre aktivitet. Fyra chimeriska proteiner konstruerades också. De skapades genom kombination av UGT72B1 och det humana enzymet UGT2B4. Dessa var alla lösliga proteiner men ingen av dem uppvisade någon aktivitet.

The interplay between lipids and membrane proteins is known to affect membrane protein topology and thus have significant effect (control) on their functions. In this PhD thesis, the influence of lipids on the membrane protein function was studied using three different membrane protein models. A monotopic membrane protein, monoglucosyldiacylglyecerol synthase (MGS) from Acholeplasma laidlawii is known to induce intracellular vesicles when expressed in Escherichia coli. The mechanism leading to this unusual phenomenon was investigated by various biochemical and biophysical techniques. The results indicated a doubling of lipid synthesis in the cell, which was triggered by the selective binding of MGS to anionic lipids. Multivariate data analysis revealed a good correlation with MGS production. Furthermore, preferential anionic lipid sequestering by MGS was shown to induce a different fatty acid modeling of E. coli membranes. The roles of specific lipid binding and the probable mechanism leading to intracellular vesicle formation were also investigated. As a second model, a MGS homolog from Synechocystis sp. PCC6803 was selected. MgdA is an integral membrane protein with multiple transmembrane helices and a unique membrane topology. The influence of different type of lipids on MgdA activity was tested with different membrane fractions of Synechocystis. Results indicated a very distinct profile compared to Acholeplasma laidlawii MGS. SQDG, an anionic lipid was found to be the species of the membrane that increased the MgdA activity 7-fold whereas two other lipids (PG and PE) had only minor effects on MgdA. Additionally, a working model of MgdA for the biosynthesis and flow of sugar lipids between Synechocystis membranes was proposed. The last model system was another integral membrane protein with a distinct structure but also a different function. The envelope stress sensor, CpxA and its interaction with E. coli membranes were studied. CpxA autophosphorylation activity was found to be positively regulated by phosphatidylethanolamine and negatively by anionic lipids. In contrast, phosphorylation of CpxR by CpxA revealed to be increased with PG but inhibited by CL. Non-bilayer lipids had a negative impact on CpxA phosphotransfer activity. Taken together, these studies provide a better understanding of the significance of the interplay of lipids and model membrane proteins discussed here.

A ciclodextrina glicosiltransferase [E.C. 2.4.1.19; CGTase] é uma enzima industrialmente importante, usada para produzir ciclodextrinas. Neste trabalho foi utilizado o planejamento experimental e a metodologia de superfície de resposta a fim de identificar as melhores condições para a produção de CGTase pela bactéria alcalófila Bacillus circulans. As melhores condições de temperatura e pH, para a produção de CGTase foram, respectivamente, 36 ºC e 9,7. Em seguida, utilizando estes parâmetros otimizados, a produção de CGTase foi avaliada em cultivos submersos, batelada e batelada alimentada, e em cultivo semi-sólido (CSS), usando um resíduo fibroso de soja (SIFR) como substrato. Nos cultivos em batelada, a atividade máxima de CGTase obtida foi de 1155 U.mL-1, em aerobiose. A produção de CGTase foi bastante influenciada pelo fluxo de ar e pela agitação, sendo que uma alta produtividade enzimática (155 U.(mLh)-1) foi obtida em condições de aeração moderada (400 rpm para a velocidade de agitação e 1,7 vvm para o fluxo de ar). Com estes parâmetros otimizados, a produtividade de CGTase obtida em cultivo em batelada alimentada foi de 137 U.(mLh)-1, com uma taxa de alimentação de 0,17 g.(Lh)-1. O crescimento celular e a síntese de CGTase, usando o resíduo fibroso de soja como substrato apresentou um rendimento de 32.776 U.g(SIFR) -1. As diferentes abordagens utilizadas neste trabalho poderão ser aplicadas para a produção de outras enzimas amilolíticas e também para a produção de CGTase com outros substratos. / Cyclodextrin glycosyltransferase [E.C. 2.4.1.19; CGTase] is an industrially important enzyme, which is used to produce cyclodextrins. In this study we report the use of experimental factorial design and response surface methodology to find the best conditions for CGTase production by alkaliphilic Bacillus circulans. The optimized calculated values for the tested variables were pH 9.7 and temperature 36oC. The CGTase production was further studied with the optimized process parameters on submerged cultivations (SC), batch or fedbatch, and solid-state cultivations (SSC) using soybean industrial fibrous residue (SIFR). The maximum CGTase activity obtained on batch cultivation was 1,155 U.mL-1 under aerobic conditions. The CGTase production was strongly affected by air flow rate and agitation speed, showing high enzyme productivity (155 U.mL-1h-1) under moderate conditions of aeration (400 rpm for speed agitation and 1.7 vvm for air flow rate). With these optimized process parameters, CGTase productivity obtained on fed-batch cultivations was 137 U.mL-1h-1, with feeding rate at 0.17 g.L-1h-1. Cell growth and CGTase synthesis in SSC using soybean industrial fibrous residue as substrate was excellent, with CGTase yield of 32,776 U.g(SIFR) -1. The different approaches used in this study may also find applications for the production of other starch-converting enzymes and also for other CGTase-producing substrates.

A ciclodextrina glicosiltransferase [E.C. 2.4.1.19; CGTase] é uma enzima industrialmente importante, usada para produzir ciclodextrinas. Neste trabalho foi utilizado o planejamento experimental e a metodologia de superfície de resposta a fim de identificar as melhores condições para a produção de CGTase pela bactéria alcalófila Bacillus circulans. As melhores condições de temperatura e pH, para a produção de CGTase foram, respectivamente, 36 ºC e 9,7. Em seguida, utilizando estes parâmetros otimizados, a produção de CGTase foi avaliada em cultivos submersos, batelada e batelada alimentada, e em cultivo semi-sólido (CSS), usando um resíduo fibroso de soja (SIFR) como substrato. Nos cultivos em batelada, a atividade máxima de CGTase obtida foi de 1155 U.mL-1, em aerobiose. A produção de CGTase foi bastante influenciada pelo fluxo de ar e pela agitação, sendo que uma alta produtividade enzimática (155 U.(mLh)-1) foi obtida em condições de aeração moderada (400 rpm para a velocidade de agitação e 1,7 vvm para o fluxo de ar). Com estes parâmetros otimizados, a produtividade de CGTase obtida em cultivo em batelada alimentada foi de 137 U.(mLh)-1, com uma taxa de alimentação de 0,17 g.(Lh)-1. O crescimento celular e a síntese de CGTase, usando o resíduo fibroso de soja como substrato apresentou um rendimento de 32.776 U.g(SIFR) -1. As diferentes abordagens utilizadas neste trabalho poderão ser aplicadas para a produção de outras enzimas amilolíticas e também para a produção de CGTase com outros substratos. / Cyclodextrin glycosyltransferase [E.C. 2.4.1.19; CGTase] is an industrially important enzyme, which is used to produce cyclodextrins. In this study we report the use of experimental factorial design and response surface methodology to find the best conditions for CGTase production by alkaliphilic Bacillus circulans. The optimized calculated values for the tested variables were pH 9.7 and temperature 36oC. The CGTase production was further studied with the optimized process parameters on submerged cultivations (SC), batch or fedbatch, and solid-state cultivations (SSC) using soybean industrial fibrous residue (SIFR). The maximum CGTase activity obtained on batch cultivation was 1,155 U.mL-1 under aerobic conditions. The CGTase production was strongly affected by air flow rate and agitation speed, showing high enzyme productivity (155 U.mL-1h-1) under moderate conditions of aeration (400 rpm for speed agitation and 1.7 vvm for air flow rate). With these optimized process parameters, CGTase productivity obtained on fed-batch cultivations was 137 U.mL-1h-1, with feeding rate at 0.17 g.L-1h-1. Cell growth and CGTase synthesis in SSC using soybean industrial fibrous residue as substrate was excellent, with CGTase yield of 32,776 U.g(SIFR) -1. The different approaches used in this study may also find applications for the production of other starch-converting enzymes and also for other CGTase-producing substrates.