As glicosidases são enzimas que apresentam uma grande variedade de enovelamentos, assim como uma alta especificidade frente a diferentes substratos. Estas enzimas têm em comum a presença de dois resíduos catalíticos, responsáveis pela clivagem das ligações glicosídicas. O uso de glicosidases nas indústrias têxtil e alimentícia, no processamento de polpa de papel e na síntese de oligossacarídeos tem incentivado a engenharia destas proteínas no sentido de melhorar suas propriedades catalíticas e estabilidade. Estudos estruturais das glicosidases têm aumentado nosso entendimento de seus mecanismos de ação catalitica, assim como dos processos de interação proteína-carboidrato. Neste trabalho apresentamos os estudos cristalográficos de duas glicosidases de fungos, sendo elas a beta-galactosidase de Penicillium sp. e a Exo-inulinase de Aspergillis awamori, assim como estudos por espalhamento de raios-X a baixos ângulos (SAXS) da beta-xylosidase de Trichoderma reesei. As estruturas cristalográficas da beta-galactosidase e de seu complexo com galactose foram determinadas pela técnica de substituição isomórfa simples com espalhamento anômalo (SIRAS) até 1.9 A angstron de resolução para a estrutura sem substrato e 2.0 angstron de resolução para o complexo. A estrutura do complexo com galactose foi usada para identificar os resíduos catalíticos, sendo o resíduo Glu 200 identificado como doador de próton e o resíduo Glu 299 como o nucleófílo. As estruturas cristalográficas da Exo-inulinase de Aspergillus awamori e de seu complexo com frutose foram também determinadas pela técnica de substituição isomórfa simples com espalhamento anômalo (SIRAS) até 1.55 angstron e 1.8 angstron de resolução, respectivamente. A partir da estrutura do complexo foi possível identificar os resíduos Asp41 e Glu241 como o nucleófilo e o doador de próton, respectivamente. Além disto, foi possível verificar que o Asp189, o qual faz parte do motivo conservado Arg-Asp-Pro (RDP), é importante no reconhecimento do substrato através de duas pontes de hidrogênio. Com o intuito de obter informações estruturais sobre a P-xylosidase seu envelope foi determinado a partir dos dados do espalhamento de raios-X a baixos ângulos. O envelope da p-xylosidase em solução foi calculado a 20 A de resolução, sendo o raio de giro e a dimensão máxima 36.9 angstron e 90 angstron, respectivamente. Usando algoritmos de reconhecimento de possíveis domínios foi determinado que esta proteína apresenta, além dos dois domínios característicos da família GHF3, um barril TIM e um domínio alfa/beta, um terceiro domínio. A predição da estrutura secundária e os dados de dicroísmo circular indicam que este terceiro domínio apresentaria um enovelamento tipo beta. / Glycosidases belong to a group of enzymes displaying a great variety of protein folds and substrate specificities. Two critically located acidic residues make up the catalytic machinery of these enzymes, responsible for the cleavage of glycosidic bonds. The applications of glycosidases in textile, food, and pulp processing, as well as in catalysts and oligosaccharide synthesis have encouraged the engineering of these proteins in order to obtain improved catalytic properties and stability. Furthermore, structural studies extend our understanding of the catalytic mechanism and the role of glycosidases in the recognition processes of their different substrates. In this work, we describe crystallographic studies of two fungi glycosidases, beta-galactosidase from Penicillium sp and Exo-inulinase from Aspergillis awamori, and the small-angle x-ray scattering (SAXS) studies of another glycosidase, beta-xylosidase (from Trichoderma reesei). The crystallographic structures of j3-galactosidase its complex with galactose were solved by single isomorphous replacement with anomalous scattering (SIRAS) using the quick cryo-soaking technique, at 1.90 angstron and 2.10 angstron resolution, respectively . The X-ray structure of the enzyme-galactose complex was useful in identifying the residue Glu 200 as the proton donor and residue Glu 299 as the nucleophile involved in catalysis. The x-ray structure of exo-inulinase and its complex with fructose were also solved by SIRAS using the quick cryo-soaking technique at 1.55 angstron and 1.8 angstron resolutions, respectively. The solved structure of the enzyme-fructose complex revealed two catalytically important residues, Asp41 and Glu241, as nucleophile and proton donor, respectively. It was also possible to see that residue Asp189, which belongs to the Arg-Asp-Pro motif, provides hydrogen bonds important for substrate recognition. In order to gain structurai insights about the beta-Xylosidase from Trichoderma reesei, we calculated their SAXS envelope. The low resolution shape of this enzyme in solution was obtained fiom synchrotron x-ray scattering data at 20 angstron resolution. The radii of gyration and the maximum dimension of the beta-Xylosidase were calculated to be 36.9 angstron and 90 angstron, respectively. In contrast to the fold of the only structurally characterized member of GHF-3, the beta-D-glucan exohydrolase, which has two distinct domains, the shape of the beta-xylosidase indicates the presence of three domains located in the same plane. Domain recognition algorithms were used to show that the C-terminal part of the mino acid sequence of the protein forms the third domain. Circular dichroism spectroscopy and secondary structure prediction programs show that this additional domain adopts predominantly the B-conformation.

No description available.

Lysosomal storage diseases are genetically inherited diseases caused by the dysfunction of lysosomal enzymes. In a normal cell, lysosomal enzymes cleave specific macromolecules as they are transported to the lysosome. However, in diseased cells, these lysosomal enzymes are either absent or malfunctioning, causing macromolecular substrates to accumulate, becoming toxic to the cell. Over fifty lysosomal storage diseases have been identified, collectively occurring in one out of 7,700 live births. We investigated the lysosomal enzyme β-galactosidase (β-gal). In order to study the biochemistry and enzymology of this protein a robust expression system was needed. The GLB1 gene has been inserted into Pichia pastoris creating high protein expressing cell lines. The result of this work will yield a high expression system for β-gal, which can then be subjected to structural and biochemical studies.

La maladie de Fabry est une maladie héréditaire de surcharge, dont la transmission est liée au chromosome X qui résulte d'un déficit de l'[alpha]-galactosidase A. Le déficit enzymatique mène à une augmentation de glycosphingolipides, notamment le globotriaosylcéramide (Gb[indice inférieur 3]), dans les tissus et fluides biologiques. Le Gb[indice inférieur 3] est donc un biomarqueur ou indicateur de la présence de cette maladie chez les patients Fabry. Nous voulions évaluer la faisabilité de procéder à un projet pilote de recherche en vue d'un dépistage néonatal urinaire de la maladie de Fabry.La variation de l'excrétion du Gb[indice inférieur 3]/créatinine urinaire chez des enfants normaux dans la période néonatale jusqu'à l'âge de 6 mois est inconnue. Cette constatation nous a conduits au questionnement suivant : existe-t-il une variation dans la quantité du Gb[indice inférieur 3]/créatinine urinaire excrétée chez des enfants normaux de 0 à 6 mois de vie? Afin de répondre à cette question, nous avons procédé à une étude permettant de doser le Gb[indice inférieur 3]/créatinine chez des enfants normaux par spectrométrie de masse en tandem et ce, en comptant sur la collaboration des parents à effectuer un total de treize prélèvements d'urine pendant une période de 6 mois. Nous avons d'ailleurs évalué ladite collaboration des parents à nous faire parvenir les échantillons d'urine de leur bébé durant cette période. Nous avons utilisé une méthode par spectrométrie de masse en tandem avec des échantillons d'urine séchée sur papier filtre pour analyser simultanément le Gb[indice inférieur 3] total urinaire et la créatinine à différents temps soit 2, 3, 4, 6, 10, 14, 21, 28 jours, de même qu'à 2, 3, 4, 5 et 6 mois chez 37 filles et 39 garçons normaux. Le traitement quantitatif des données de la créatinine et du Gb[indice inférieur 3] urinaire a été fait par le logiciel QuanLynx (Waters). Nous avons divisé la variable du temps en quatre périodes pour les fins d'analyses statistiques : (1) < 6 jours; (2) 6-29 jours; (3) 30-90 jours; (4) > 90 jours. Nous avons procédé à des analyses statistiques comparatives du rapport Gb[indice inférieur 3]/créatinine de 728 échantillons pour les deux cohortes en fonction du temps. Une analyse de variance a été faite pour évaluer l'effet de l'âge et du sexe sur le rapport du logarithme du Gb[indice inférieur 3]/créatinine urinaire et l'effet de l'âge et du sexe sur la quantité d'excrétion de la créatinine urinaire seulement. Nous avons observé un effet significatif de l'âge sur la créatinine (p < 0.0001). En ce qui concerne les résultats du rapport du Gb[indice inférieur 3]/créatinine, il y a une augmentation non significative de la médiane dans les périodes 1 et 2 pour les garçons (Période 1: Médiane 53.9; Min-Max 0 - 369.3 [micro]g/mmol créatinine; Période 2: Médiane 92.5; Min-Max 0 - 611.1 [micro]g/mmol créatinine ; p = 1.0000). Chez les filles, l'excrétion du Gb[indice inférieur 3]/créatinine est plus élevé à la naissance et présente une tendance à l'accroissement entre les périodes 1 et 2 (Période 1: Médiane 59.5; Min-Max 0 - 669.9 [micro]g/mmol créatinine; Période 2: Médiane 96.1; Min-Max 0 - 456.1 [micro]g/mmol créatinine ; p = 1.0000). Par ailleurs, l'excrétion du Gb[indice inférieur 3]/créatinine chez les garçons diminue de façon significative entre les périodes 2 et 4 (Période 2: Médiane 92.5; Min-Max 0 - 611.1 [micro]g/mmol créatinine; Période 4: Médiane 14.6; Min-Max 0 - 158.5 [micro]g/mmol créatinine ; p < 0.0001); au niveau des filles, il y a une diminution non significative de la médiane de la période 2 à la période 3 (Période 2 : Médiane 96.1; Min-Max 0 - 456.1 [micro]g/mmol créatinine ; Période 3 : Médiane 35.6; Min-Max 0 - 254.4 [micro]g/mmol créatinine p = 0.2290) et une légère augmentation à la période 4 (Période 4 : Médiane 42.7; Min-Max 0 - 617.2 [micro]g/mmol créatinine). Ainsi, nous pouvons constater qu'il existe une grande variabilité de l'excrétion du Gb[indice inférieur 3]

<p>The two techniques aqueoustwo-phase partitioning and expanded bed adsorption that bothare suitable for primary protein recovery were studied. Most ofthe work was focused on partition in aqueous two-phase systemsand in particular on the possibility to effect the partitionbehaviour by fusion of short peptide tags or protein domains tothe target protein.</p><p>The partitioning of fusionproteins between different variants of the domain tag Z and thenaturally occurring protein DNA Klenow polymerase were studiedin Breox/Reppal aqueous two-phase systems. Most studies wereperformed with cell homogenate. The Breox/Reppal system was infocus because if the fusion protein can be partitioned to theBreox-rich top phase the next step can be a thermoseparatingaqueous two-phase system. When the Breox phase is heated to50°C it switches from a one-phase system to a two-phasesystem resulting in an almost pure water rich top phase andhighly concentrated Breox-rich bottom phase. The Breox can thenbe reused and the protein recovered from the water phase. TheZ-domain was genetically modified in different ways to Z_basic1, Z_acid2and Z_trp12and fused to the Klenow protein to try toenhance partitioning to the Breox-rich phase. From theexperiments it was not possible to observe any effects on thepartition behaviour irrespectively of tested properties of thedomain tag. Despite the absence of domain tag effects highK-values, i.e. partition to the Breox-rich top phase, wereobserved in the Breox/Reppal system. However, the proteinK-values seemed to be rather sensitive to the cell homogenateload and showed a tendency to decrease with increased cellhomogenate load. Also increased phosphate concentration reducedthe K-values. The partitioning of cell debris also seemed todependent on the cell homogenate load. At higher homogenateload (<=20g DW/L) clear Breox-rich top phases were observedwith the cell debris collected in Reppal-rich bottomphases.</p><p>Two different tetrapeptides,AlaTrpTrpPro and AlaIleIlePro were inserted near the C-terminusof the protein ZZT0. The Trp-rich peptide unit stronglyincreased both the partitioning of ZZT0 into the poly(ethyleneglycol) (PEG)-rich phase in a PEG/potassium phosphate aqueoustwo-phase system and its retention on PEG and propylhydrophobic interaction chromatographic columns with potassiumphosphate as eluent in isocratic systems. Both the partitioningand the retention increased with increasing number of Trp-richpeptide units inserted into ZZT0. Insertion of Ile-richtetrapeptide units affected the partitioning and retention to amuch lesser extent. Partition and modelling data also indicateda folding of inserted Trp and Ile tetrapeptide units, probablyto minimise their water contact. It was also investigated howto predict the partitioning of proteins in isoelectricPEG/phosphate aqueous two-phase systems.</p><p>The capture ofß-galactosidase from<i>E. coli</i>cell homogentate (50g DW/L) by metal chelatexpanded bed adsorption was studied. These experiments showedthat capture, with a certain degree of selectivity, andclarification of ß-galactosidase could be achieved from acell homogenate. However, a rather low recovery of about 35 %was obtained at a capacity of 0.25mg/mL of gel. Thus, severalparameters remain to be optimised like the load buffercomposition and the cell homogenate load.</p><p><b>Keywords:</b><i>E. coli</i>, aqueous two-phase systems, fusion proteins,hydrophobic interaction chromatography, expanded bedadsorption, ß-galactosidase, Klenow polymerase, Z-domain,peptide tags</p>

<p>Valorizacija permeata kao sporednog proizvoda industrije mleka je od izuzetnog ekolo&scaron;kog, ekonomskog i tehnolo&scaron;kog značaja.<br />Cilj doktorske disertacije je razvoj tehnolo&scaron;kog procesa prerade permeata, kao sporednog proizvoda dobijenog nakon ultrafiltracije mleka tokom proizvodnje feta sira i svežeg (&bdquo;mladog&ldquo;) sira. Ispitana je mogućnost enzimske hidrolize laktoze u permeatu kori&scaron;ćenjem enzima &beta;-galaktozidaze izolovanog iz Kluyveromyces lactis u koncentraciji 0,1, 0,3 i 0,5 g/100g na temperaturama 20&ordm;, 30&ordm; i 40 &ordm;C. Praćene su promene sadržaja laktoze, D&ndash;galaktoze i D&ndash;glukoze u vremenskim intervalima tokom 60 minuta. Posebna faza istraživanja obuhvatila je matematičko modelovanje i kinetiku procesa hidrolize laktoze u permeatu pod dejstvom &beta; &ndash;galaktozidaze i primenu hidrolizovanog permeata u proizvodnji mlečnih napitaka po odabranoj formulaciji. Predložen je tehnolo&scaron;ki proces proizvodnje napitka na bazi hidrolizovanog permeata sa dodatkom voćnih baza. Utvrđeni su parametri kvaliteta i trajnosti napitaka tokom 60 dana skladi&scaron;tenja.<br />Na temperaturi 40&deg;C dodatkom enzima &beta; -galaktozidaze u koncentraciji 0,1g/100g za 60 minuta postiže se 100% stepen hidrolize prisutne laktoze u permeatu. Sa većom koncentracijom enzima, 0,3 g/100g odnosno 0,5g/100g, na istoj temperaturi, isti efekat se postiže za 20 minuta.<br />Ispitivanjem kinetike hidrolize laktoze potvrđena je kinetika prvog reda. Generalno posmatrano visoki koeficijenti determinacije pokazuju dobro poklapanje eksperimentalnih rezultata i matematičkog modela reakcije prvog reda. Vrednosti se kreću od 0,974 (temperatura 20&deg;C) do preko 0,990 (na temperaturama 30&deg;C i 40&deg;C) pri koncentraciji enzima 0,1g/100g.<br />Proizvedeni napici od hidrolizovanog permeata su delaktozirani i ne sadrže mlečnu mast. Od ukupnih &scaron;ećera u svim napicima vi&scaron;e od 50% čini glukoza: 50,16% - napitak &scaron;umsko voće, 50,42% - napitak pomorandža/&scaron;argarepa, 54,65% - napitak multivitamin, odnosno 55,13% - napitak crveno voće.<br />Najveći sadržaj vitamina C nakon proizvodnje imao je napitak sa dodatkom voćne baze multivitamin 0,3972 mg/100g, zatim &scaron;umsko voće 0,2887 mg/100g i pomo-randža/&scaron;argarepa 0,1999 mg/100g.<br />Najveću vrednost antioksidativne aktivnosti nakon proizvodnje pokazali su uzorci napitka sa multivitaminom i &scaron;umskim voćem. Tokom perioda skladi&scaron;tenja dolazi do smanjenja DPPH vrednosti. Najmanji pad je u napitku sa pomorandžom / &scaron;argarepom (smanjenje za 17%), a najveći u napitku sa &scaron;umskim voćem (za 39%). Analizirani uzorci sadrže ukupnih polifenola u intervalu od 47,84 do 120,38 mg GAE/l u zavisnosti od vrste napitka, odnosno dodatih voćnih baza.<br />Generalno može se zaključiti da se prime-njenim tehnolo&scaron;kim procesom dobijaju napici stabilnog fizičko-hemijskog sastava tokom 60 dana skladi&scaron;tenja, visoke nutritivne i niske energetske vrednosti.</p> / <p>Valuation of the permeate as a by-product of the dairy industry is of great ecological, economic and technological importance.<br />The aim of the PhD thesis is the development of the technological process of refining permeate, as a by-product obtained after ultrafiltration of milk during the production of feta cheese and fresh cheese. The possibility of enzymatic hydrolysis of the lactose in the permeate using the enzyme &beta;-galactosidase isolated from Kluyveromyces lactis in a concentration of 0.1, 0.3 and 0.5 g / 100 g at a temperature of 20&deg;, 30&deg; and 40&deg; C was examined. Changes in the content of lactose, D-galactose and D-glucose at intervals of 60 minutes were monitored. A special stage of the research included mathematical modeling and kinetics of lactose hydrolysis in the permeate under the influence of &beta;-galactosidase and application of hydrolyzed permeate in the production of dairy products under the selected formulation. A technological process of producing a beverage on the basis of hydrolyzed permeate with the addition of fruit bases was suggested. Quality and durability parameters were determined for drinks during the 60 days of storage.<br />Addition of the enzyme &beta;-galactosidase at a concentration of 0.1 g / 100 g for 60 minutes at a temperature of 40 &deg; C a 100% degree of hydrolysis of lactose is achieved, present in the permeate. With a higher concentration of enzyme, 0.3 g / 100 g or 0.5 g / 100g, at the same temperature, the same effect can be achieved in 20 minutes.<br />By examining the kinetics of lactose hydrolysis the first order kinetics was confirmed. Generally high coefficients of determination show good correspondence between the experimental results and the mathematical model of the first order reaction. Values range from 0.974 (at a temperature of 20&deg; C) up to over 0.990 (at temperatures 30&deg; C and 40&deg; C) at a an enzyme concentration of 0.1g / 100g.<br />Beverages produced from hydrolyzed permeate are lactose-free and fat-free products. More than half of the total sugar content in all beverages consists of glucose: 50.16%-forest fruit beverage, 50.42%-beverage orange/carrot, 54.65% beverage multivitamin and 55.13% - beverage red fruit.<br />The highest vitamin C content after production was in a beverage with the addition of fruit base multivitamin (0.3972 mg/100g), followed by forest fruit (0.2887 mg/100g) and orange/carrot (0.1999 mg/100g).<br />Beverage samples with multivitamin and forest fruits showed the highest value of antioxidant activity after production. During the storage period there is a reduction of DPPH values. The smallest decrease was in the beverage with orange/carrot (decreased 17%), and the biggest in the beverage with forest fruit (39%). The content of polyphenols in analyzed samples ranges from 47.84 to 120.38 mg GAE/L depending on the type of beverage and added fruit base.<br />Overall it can be concluded that the applied technological process gives beverages of stable physical and chemical content during the 60 days of storage, of high nutritional value and low energy.</p>

The marked increase in surface-to-volume ratio associated with microscale devices for hemodialysis leads to problems with hemocompatibility and blood flow distribution that are more challenging to manage than those encountered at the conventional scale. In this work, stable surface modifications with pendant polyethylene oxide (PEO) chains were produced on polycarbonate microchannel and polyacrylonitrile membrane materials used in construction of microchannel hemodialyzer test articles. These coatings were evaluated in relation to protein repulsion, impact on urea permeability through the membrane, and impact on bubble retention through single-channel test articles. PEO layers were prepared by radiolytic grafting of PEO-PBD-PEO (PBD = polybutadiene) triblock copolymers to microchannel and membrane materials. Protein adsorption was detected by measurement of surface-bound enzyme activity following contact of uncoated and PEO-coated surfaces with ��-galactosidase. Protein adsorption was decreased on PEO-coated polycarbonate and polydimethyl siloxane (PDMS) materials by 80% when compared to the level recorded on uncoated materials. Protein adsorption on membrane materials was not decreased with PEO-PBD-PEO treatment; a PEI (polyethylene imide) layer exists on the AN69 ST membrane which is intended to trap heparin during membrane pre-treatment. It is still unclear how this PEI layer interacts with PEO-PBD-PEO. Neither the PEO-PBD-PEO triblocks nor the irradiation process was observed to have any effect on polyacrylonitrile membrane permeability to urea, nor did the presence of additional fibrinogen and bovine serum albumin (BSA) in the urea filtrate. The PEO-PBD-PEO treatment was not able to visibly reduce bubble retention during flow through single-channel polycarbonate test articles, however, the rough surfaces of the laser-etched polycarbonate microchannels may be causing this bubble retention. This surface treatment holds promise as a means for imparting safe, efficacious coatings to blood processing equipment that ensure good hemocompatibility and blood flow distribution, with no adverse effects on mass transfer. / Graduation date: 2013

&alpha / -Galactosidase is an exo-glycosidase that hydrolyses non-reducing, &alpha / -1,6-linked &alpha / -galactose units from oligosaccharides, galactomannans, and galactolipids. &alpha / -Galactosidase activity has biotechnological, industrial, and medical importance. &alpha / -Galactosidase from A. fumigatus IMI 385708, in particular, can catalyse unique hydrolysis and transgalactosylation reactions on polymeric substrates. In this study, &alpha / -galactosidase of the human pathogen A. fumigatus IMI 385708 was first produced in a GRAS organism, Aspergillus sojae. For this aim, &alpha / -galactosidase gene (aglB) of A. fumigatus IMI 385708 was ligated onto pAN52-4 vector (Acc. No: Z32699) and transformed into Aspergillus sojae ATCC11906, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (gpdA) of A. nidulans and the signal sequence of glucoamylase gene (glaA) of A. niger. This allowed high level of &alpha / -galactosidase production on glucose instead of locust bean gum (2.45 U/mL), corresponding to a 3-fold increase in volumetric production. Next, using response surface methodology, carbon and nitrogen sources and agitation speed were optimized (10.5% molasses (w/v) / 1.3% NH4NO3 (w/v) / 276 rpm). Compared to non-optimized cultivation, a further 4-fold increase in &alpha / -galactosidase production (10.4 U/mL) was achieved. Recombinant &alpha / -galactosidase was purified 18.7-fold using Anion Exchange and Hydrophobic Interaction Chromatography with an overall yield of 56% and 64.7 U/mg protein. The Vmax and Km values for the hydrolysis of p-nitrophenyl &alpha / -D-galactopyranoside were 78 U/mg protein and 0.45 mM, respectively. Optimum pH and temperature for &alpha / -galactosidase activity were between pH 4&ndash / 6 and 50&ndash / 60 &deg / C, respectively. Among the tested chemical agents, Ag+, Hg2+, and Fe2+ drastically decreased the activity, while biotin, I+1, Mn+2, Pb+2, Li+1, and Mg+2 enhanced between 12&ndash / 29%. To analyse the influence of osmotic stress as a means of further inducing &alpha / -galactosidase production, salt was added into the complete growth medium. In addition to enzyme production, fungal growth and morphology were analysed for both &lsquo / salt-adapted&rsquo / and &lsquo / salt non-adapted&rsquo / A. sojae Ta1 cells in the presence of KCl, MgCl2, MgSO4, NaCl, and Na2SO4 at 1 M and 2 M. Accordingly, 3-fold increase in &alpha / -galactosidase production was achieved by non-adapted cells in the presence of 1 M NaCl. Exposure of A. sojae Ta1 cells to salt resulted in predominantly mycelial form, rather than the pellet form observed under normal conditions. Finally, the transgalactosylation ability of &alpha / -galactosidase was studied. &alpha / -Galactosidase efficiently catalysed galactose transfer to different monosaccharides and disaccharides in the presence of pNP&alpha / Gal as monitored by TLC, ESI-MS, and HPLC.

Crystallization is often a major bottleneck to macromolecular structure determination. This is particularly true for membrane proteins, which have hydrophobic surfaces that cannot readily form crystal contacts. Of the roughly 109,000 protein structures in the PDB, only about 539 represent unique membrane proteins, despite immense interest in membrane proteins from both a biological and therapeutic standpoint. Membrane protein crystallization has been facilitated by the development of new detergents, lipidic cubic phase methods, soluble protein chimeras, and non-covalent protein complexes. The design process of protein fusion constructs and non-covalent antibody fragments specific for each target membrane protein, however, is costly and time-consuming. An improved, more general method of membrane protein co-crystallization is needed. This dissertation details the development of two approaches for cost-effective non-covalent crystallization chaperones: (1) Engineered hypercrystallizable Fab antibody fragment with high affinity for EYMPME (EE epitope), which form complexes with EE-tagged soluble and membrane proteins. (2) Engineered monomeric streptavidin (mSA2) for complexation with biotinylated membrane proteins. Both methods are generalizable through insertion of a short epitope into a surface-exposed loop of a membrane protein by site directed mutagenesis. Crystallization trials of representative chaperone-membrane protein complexes and possible difficulties with the approach are discussed.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Immobilized enzymes provide many advantages when compared to the usage of their free forms. Among these ones, remarkable advantages are the possibility of the biocatalyst reusability, easy separation at the end of the process, its usage in continuous way and the enhancement of its stability. This work was performed aiming the immobilization of the &#945;-galactosidase enzyme from Aspergillus niger in ion exchange resin and the evaluation of its catalytic activity. Firstly, tests were performed in five different resins: Amberlite 252-Na, Dowex Marathon A, Dowex Marathon C, Duolite A-568 e Duolite S-761. According to the results, Duolite A-568 was chosen as the best support. Therefore, studies were done aiming the optimization of the immobilization process in this resin. Glutaraldehyde 1% (v/v) was used before the enzyme adsorption process and it enhanced the operational stability of the immobilized enzyme. Preliminary tests did not showed difference for the immobilization process at the temperatures of 25 and 40°C. A full factorial design and a central composite design were performed to study the best immobilization conditions varying the pH, the &#945;-galactosidase concentration and the immobilization time. The results led to use the following immobilization conditions: pH 4.5; 15 g/L of &#945;-galactosidase and 3 hours of immobilization. The temperature of maximum activity occurred at 60°C for both free and immobilized enzyme. The activation energy calculated by linear adjustment of Arrhenius equation was 5.66 kcal/mol for soluble &#945;-galactosidase and 4.48 kcal/mol for immobilized &#945;-galactosidase. The optimum pH range obtained for free enzyme was 4.0-5.0 and for immobilized enzyme it was 3.0-6.0. The immobilization process improved the &#945;-galactosidase activity in alkaline pHs. Analysis of pH stability showed that both forms of enzyme were resistant for the pH ranges studied (3.5 to 7.5 for free and 3.0 to 8.0 for immobilized). However, the thermal stability of the biocatalyst immobilized in the support decreased. The kinetic studies without inhibition showed closed values of maximum speed (Vmax) for both enzyme forms (194.5 U for free and 187.7 U for immobilized). Although, the Michaelis-Menten constant (Km) of immobilized enzyme was higher than the free one (18.8 and 12.5 g/L, respectively). The hydrolysis reaction of raffinose was inhibited by the addition of the reaction products, sucrose and galactose, and the results of inhibition by galactose pointed for the competitive inhibition type. Then, storage tests of immobilized &#945;-galactosidase showed that the enzyme maintained its activity even after 145 days when kept at the temperature of 4°C. / O uso de enzimas imobilizadas proporciona muitas vantagens em relação ao seu uso na forma livre. Dentre estas vantagens se destacam a possibilidade de reutilização do biocatalisador, a sua fácil separação ao final do processo, a utilização em modo contínuo e o aumento de sua estabilidade. Este trabalho foi desenvolvido com o objetivo de imobilizar a enzima &#945;-galactosidase de Aspergillus niger em resina de troca iônica e avaliar a sua atividade catalítica. Inicialmente, foram feitos testes preliminares de imobilização em 5 tipos de resinas: Amberlite 252-Na, Dowex Marathon A, Dowex Marathon C, Duolite A-568 e Duolite S-761. Pelos resultados obtidos, Duolite A-568 foi selecionada como melhor suporte e, portanto, estudos foram feitos para a otimização do processo de imobilização nesta resina. Glutaraldeído na concentração de 1% (v/v) foi utilizado anteriormente ao processo de adsorção da enzima e melhorou a estabilidade operacional da &#945;-galactosidase imobilizada. Testes preliminares não indicaram diferença do processo de imobilização para temperaturas de 25 e 40°C. Realizou-se um planejamento fatorial completo e um planejamento composto central para estudar as melhores condições de imobilização variando-se o pH, concentração de &#945;-galactosidase e tempo de imobilização. Os resultados obtidos levaram a utilizar as seguintes condições de imobilização: pH 4,5, concentração de &#945;-galactosidase de 15 g/L e tempo de imobilização de 3 horas. A temperatura de máxima atividade enzimática foi 60°C tanto para a enzima livre quanto imobilizada. O valor da energia de ativação encontrado pelo ajuste linear da equação de Arrhenius foi de 5,66 kcal/mol para &#945;-galactosidase solúvel e 4,48 kcal/mol para &#945;-galactosidase imobilizada. A faixa de pH ótimo obtido para a enzima livre foi 4,0-6,0 e para a enzima imobilizada foi 3,0-6,0. O processo de imobilização melhorou a atividade da &#945;-galactosidase para pHs mais alcalinos. A análise de resistência ao pH mostrou que ambas as formas da enzima foram resistentes para as faixas estudadas (3,5 a 7,5 para livre e 3,0 a 8,0 para imobilizada). No entanto, a resistência térmica do biocatalisador retido no suporte foi menor. O estudo cinético sem inibição apresentou valores de velocidade máxima (Vmáx) próximos para as duas formas da &#945;-galactosidase (194,5 U para livre e 187,7 U para imobilizada), porém o Km da forma imobilizada foi maior que o da livre (18,8 g/L e 12, 5 g/L de rafinose, respectivamente). A reação de hidrólise da rafinose foi inibida pela adição dos produtos da reação, sacarose e galactose, sendo que os resultados de inibição por galactose apontam para o tipo de inibição competitiva Por fim, testes de estocagem da &#945;-galactosidase imobilizada mostraram que a enzima manteve sua atividade mesmo após 145 dias mantida a temperatura de 4°C. / Mestre em Engenharia Química