Submitted in fulfilment of the requirements of the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2001. / Kluyveromyces sp. have adapted to existence in milk due to the evolution of permeabilisation and hydrolytic systems that allow the utilisation of lactose, the sugar most abundant in milk. Lactose hydrolysis, to equimolar units of glucose and galactose, is facilitated by a glycoside hydrolase, i.e., β-galactosidase (EC 3.2.1.23). The versatility of this enzyme allows its application in numerous industrial processes, amongst the most significant of which, is its role in the alleviation of lactose intolerance, one of the most prevalent digestive ailments, globally. In this study, β-galactosidase production by Kluyveromyces lactis UOFS y-0939 was initially optimised in shake flask culture with lactose as the sole carbon source, and thereafter, production was scaled up to batch, fedbatch and continuous culture. Shake flask studies revealed optimum conditions of 30°C, pH 7 and a 10% inoculum ratio, to be most favourable for β-galactosidase synthesis, producing a maximum of 0.35 ± 0.05 U.ml-1 when cell lysates were prepared by ultrasonication with glass beads. Batch cultivation in 28.2 and 40 g.L-1 lactose revealed that elevated levels of the carbon source was not inhibitory to β-galactosidase production, as maximum enzyme activities of 1.58 and 4.08 U.ml-1, respectively, were achieved. Cell lysates prepared by ultrasonication and homogenisation were compared and homogenised cell lysates were more than 3.5 fold higher that those prepared by ultrasonication, proving homogenisation to be the superior method for cell disruption. The lactose feed rate of 4 g.L-1.h-1 in fed-batch culture operated at ± 20.4% DO, appeared to be inhibitory to biomass production, as indicated by the lower biomass productivity in fed-batch (0.82 g.L-1.h-1) than batch culture (1.27 g.L-1.h-1). Enzyme titres, however, were favoured by the low DO levels as a maximum of 8.7 U.ml-1, 5.5 fold more than that obtained in batch culture, was achieved, and would be expected to increase proportionally with the biomass. Continuous culture operated at a dilution rate of 0.2 h-1, under strictly aerobic conditions, revealed these conditions to be inhibitory to the lactose consumption rate, however, the non-limiting lactose and high DO environment was favourable for β-galactosidase synthesis, achieving an average of 8 ± 0.9 U.ml-1 in steady state.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: ß-Galactosidase of Escherichia coli is the equivalent of lactase in humans and has the ability to bind and hydrolyse lactose. Lactase de ciency is a common phenomenon present in almost 70% of the world's population. This has resulted in greater than before demands on the food processing industry to develop a method that will allow for the hydrolysis of the disaccharide lactose in milk but will also allow for the removal of the remaining active enzyme. In this thesis, a new method, that is bio-speci c and well characterized for the removal of lactose from a lactose containing solution, is described. The E537D mutated version of ß-Galactosidase, which has a much lower activity compared to the wildtype and is able to bio-speci cally bind lactose for longer periods, was covalently immobilised to commercially available magnetic nanoparticles (fl uidMAG-Amine) via two coupling strategies. Glutaraldehyde is a cross-linking agent that reacts with amine groups, while N- (3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) is a coupling agent that activates carboxylic groups. These agents are widely used for the coupling of biomolecules to solid supports. The covalently coupled fluidMAG-E537D ß-Galactosidase particles were characterized regarding retained enzymatic activity and ability to bind and physically remove lactose from a lactose containing solution by applying an external magnetic eld, after lactose binding, to the enzyme-particle complex in solution. Each component aimed at yielding this functionally immobilised enzyme complex was studied and optimized to contribute to the development of this novel technique, which is a ordable and simple, for the removal of lactose from solution for the ultimate production of lactose free milk. Results indicated the glutaraldehyde method of ß-Gal cross-linking to fluidMAG-Amine to be the preferred strategy since it allowed an increased carrier capacity of protein to the particles. The glutaraldehyde cross-linked protein also exhibited a two-fold higher activity than the EDC coupled protein. Furthermore, the glutaraldehyde cross-linked fluidMAG-E537D ß-Gal was able to physically remove 34 % of the lactose from a 0.2 nmol/L lactose in solution. This, therefore, con rmed the potential use of this novel technique in the food processing industry. / AFRIKAANSE OPSOMMING: ß-Galaktosidase vanaf Escherichia coli is dieselfde as laktase in mense en beskik oor die vermoë om laktose te bind en te hidroliseer. 'n Gebrek aan laktase kom algemeen voor en ongeveer 70 % van die wêreldbevolking ly hieraan. Laasgenoemde het daartoe gelei dat daar meer druk as vantevore op die voedselproduksie industrie is om 'n metode te ontwikkel waarmee die hidrolise van die disakkaried laktose in melk moontlik sal wees asook die verwydering van die oorblywende aktiewe ensiem. In hierdie tesis word 'n nuwe metode beskryf wat biospesi ek en goed gekarakteriseer is vir die verwydering van laktose vanuit 'n laktose bevattende oplossing. Die E537D gemuteerde weergawe van ß-Galaktosidase, wat beskik oor 'n baie laer aktiwiteit as die wildetipe asook die vermoë om laktose biospesi ek vir langer periodes te bind, is kovalent geïmmobiliseer op kommersieel beskikbare magnetiese nanopartikels (fluidMAG-Amine) via twee koppelingsstrategieë. Glutaraldehied is 'n kruisbindingsagent wat met amino groepe reageer, terwyl EDC 'n koppelingsagent is wat karboksie groepe aktiveer. Hierdie agente word algemeen gebruik vir die binding van biomolekules aan soliede matrikse. Die kovalent gekoppelde fluidMAG-E537D ß-Galaktosidase partikels is gekarakteriseer met betrekking tot behoue ensimatiese aktiwiteit en vermoë om laktose te bind en sies te verwyder vanuit 'n oplossing wat laktose bevat deur 'n eksterne magneetveld op die ensiem-partikel kompleks in oplossing toe te pas, nadat die binding van laktose plaasgevind het. Elke komponent van hierdie funksioneel geïmmobiliseerde ensiemkomplekse is ondersoek en geoptimaliseer met die doel om by te dra tot die ontwikkeling van 'n nuwe tegniek wat bekostigbaar en eenvoudig is vir die verwydering van laktose vanuit 'n oplossing vir die uiteindelike gebruik in die produksie van laktose-vrye melk. Resultate het getoon dat die glutaraldehied metode van ß-Gal kruisbinding op fluidMAG-Amine verkies word aangesien dit 'n verhoogde draerkapasiteit van proteïene op die partikels moontlik maak. Die glutaraldehied gekoppelde proteïene beskik ook oor twee keer meer aktiwiteit as die EDC gekoppelde proteïene. Die glutaraldehied gekoppelde fluidMAG-E537D ß -Gal kon 34 % van die laktose teenwoordig in 'n 0.2 nmol/L laktose oplossing sies verwyder. Hierdie het dus die potensiële gebruik van hierdie nuwe metode in die voedselproduksie industrie bevestig.

Single enzymes of E. coli sourced B-galactosidase were analysed in effort to expand the wealth of knowledge in the area of heterogeneity. Static and dynamic heterogeneity was studied with respect to catalytic rate, electrophoretic mobility, and heat shock protein chaperone systems. Temperature was found to be a contributing factor to the observed range of dynamic heterogeneity, with the range increasing with temperature. The inhibitor dissociation constant was determined to be a heterogeneous property of B-galactosidase. A novel assay was developed in which a single enzyme molecule was subjected to three separate solutions while the enzyme itself remained free in solution. / October 2016

A β-galactosidase (E.C 3.2.1.23) é uma das enzimas mais empregadas na indústria de alimentos sendo utilizada na hidrólise da lactose. Neste trabalho foram utilizadas duas metodologias para imobilização desta enzima. Na primeira delas foi empregado como suporte um material híbrido à base de sílica que possui um grupo orgânico catiônico covalentemente ligado. A adsorção da enzima a este material apresentou eficiência que variou de 74 a 53% com o aumento da quantidade de enzima aplicada ao suporte. A baixa estabilidade térmica da enzima imobilizada obtida e as prováveis fracas interações envolvidas na sua adsorção a este suporte podem explicar o decréscimo de atividade observada durante as sucessivas bateladas de hidrólise da lactose. Na primeira batelada o grau de hidrólise foi de 90,9% e no final da última batelada (4ª), a enzima foi capaz de converter apenas 13% do substrato. A segunda metodologia utilizada foi imobilização covalente da enzima em um filme de celulose/líquido iônico modificado com uma poliamina e ativado com glutaraldeído. A presença da poliamina foi confirmada por análises de infravermelho. Após a imobilização, a enzima reteve 60% de sua atividade inicial. Bons resultados de hidrólise da lactose em batelada foram obtidos tanto a 7ºC como a 35ºC e foi possível reutilizar a enzima imobilizada por 16 ciclos consecutivos, a 7ºC, sem mudanças significativas na atividade enzimática. O valor de Km para a enzima imobilizada no material híbrido à base de sílica foi de 9,17 mM e para a enzima imobilizada nos filmes de celulose foi de 11,22 mM, ambos apresentaram um acréscimo quando comparados ao Km enzima livre (1,25 mM), devido à dificuldade de acesso do substrato ao sítio ativo da enzima. Não houve mudança no pH e temperatura ótimos da enzima imobilizada em relação à enzima livre em nenhum dos métodos testados. / β-galactosidase (E.C 3.2.1.23) is the most widely used enzymes in the food industry and its employed in the lactose hydrolysis process. In this study, two methodologies were used to test their immobilization. In the first, the enzyme was immobilized by adsorption in one silica based hybrid material that contains a cationic organic group covalently linked. The efficiency of immobilization showed a decrease of 74 to 53% by increasing the protein load applied to the support. The low thermo stability of the immobilized enzyme and the probable weak interactions involved in their adsorption, could explain the decrease in enzyme activity observed in the successive batch hydrolysis of lactose. In the first run, the degree of lactose hydrolysis was 90.9% and, at the end of the last run (4th), the enzyme was able to convert only 13% of the substrate. The second methodology used was the covalent immobilization of the enzyme on a cellulose/ionic liquid film, modified with a polyamine and activated using glutaraldehyde. The presence of a polyamine was confirmed by infrared analysis. After immobilization, the enzyme retained 60% of its initial activity. Highly efficient lactose conversion was achieved in a batch process at 7ºC and 35ºC and was possible to reuse the immobilized enzyme in 16 repeated cycles, at 7ºC, without any drastic decrease in enzyme activity. Km value for the immobilized enzyme in silica based hybrid material was 9.17 mM and for the enzyme immobilized in the film of cellulose/ionic liquid was 11.22 mM, both showing an increase compared with the Km value for free enzyme (1.25 mM), due to the difficulty of access of the substrate to the active sites of the enzyme. The immobilized enzyme did not show any changes in the optimal pH and temperature when compared to the free enzyme in both methods tested.

Fabry disease (FD) is an X-linked lysosomal storage disorder with intracellular accumulation of globotriaosylceramide (Gb3) due to α-galactosidase A deficiency. We studied α-galactosidase A knockout mice (GLA KO) as a model for sensory disturbance and pain in FD. Pain associated behavior of young (3 months) and old (≥18 months) GLA KO mice and wildtype (WT) littermates in an inflammatory and a neuropathic pain model was investigated. Furthermore, affective and cognitive behavior was assessed in the naïve state and in an inflammatory pain model. Gene and protein expression of pain associated ion channels and Gb3 accumulation in dorsal root ganglion (DRG) neurons was determined. We also performed patch clamp analysis on cultivated DRG neurons and human embryonic kidney 293 (HEK) cells expressing voltage-gated-sodium channel 1.7 (Nav1.7) as an in vitro model of FD. Intracellular Gb3 deposits were modulated using shRNA silencing of α-galactosidase A. After intraplantar injection of complete Freund`s adjuvant (CFA) and chronic constriction injury (CCI) of the right sciatic nerve, old GLA KO mice did not develop heat and mechanical hypersensitivity in contrast to young GLA KO and old WT mice. Additionally, we found no relevant differences between genotypes and age-groups in affective and cognitive behavior in the naïve state and after CFA injection. Gene and protein expression analysis provided no explanation for the observed sensory impairment. However, cultured DRG neurons of old GLA KO mice revealed a marked decrease of sodium and Ih-currents compared to young GLA KO and old WT mice. DRG neurons of old GLA KO mice displayed substantial intracellular accumulation of Gb3 compared to young GLA KO and old WT mice. Similar to cultured neurons, sodium currents were also decreased in HEK cells treated with shRNA and consecutively increased intracellular Gb3 deposits compared to the control condition, but could be rescued by treatment with agalsidase-alpha. Our study unveils that, similar to patients with FD, GLA KO mice display age-dependent sensory deficits. However, contrary to patients, GLA KO mice are also protected from hypersensitivity induced by inflammation and nerve lesion due to Gb3-dependent and reversible reduction of neuronal sodium- and Ih-currents. Our data provide evidence for direct Gb3-dependent ion channel impairment in sensory DRG neurons as a potential contributor to sensory dysfunction and pain in FD. / Bei Morbus Fabry (M. Fabry) handelt es sich um eine X-chromosomal vererbte, lysosomale Speichererkrankung mit intrazellulärer Akkumulation von Globo-triaosylceramid (Gb3) aufgrund eines α-Galaktosidase-A Mangels. Um die Pathophysiologie des M. Fabry aufzuklären, untersuchten wir die α-Galaktosidase-A defiziente Maus (GLA KO) als Modell für sensible Wahrnehmungsstörungen und Schmerz. Das schmerzassoziierte Verhalten von jungen (3 Monate) und alten (≥18 Monate) GLA KO Mäusen und Wildtyp (WT) Wurfgeschwistern wurde in einem Entzündungs- und einem neuropathischen Schmerzmodell untersucht. Zudem wurde das affektive und kognitive Verhalten im naiven Zustand und in einem Entzündungsschmerzmodell betrachtet. Auf molekularer Ebene wurden die Gen- und Proteinexpression von schmerzassoziierten Ionenkanälen und die Gb3-Akkumulation in Spinalganglionneuronen (dorsal root ganglion, DRG) bestimmt. Darüber hinaus wurden kultivierte DRG Neurone und humane embryonale Nierenzellen 293 (HEK) mittels Patch-clamp-Analyse elektrophysiologisch untersucht. Die HEK Zellen dienten als in vitro Modell für M. Fabry und exprimierten stabil den spannungsgesteuerten Natriumkanal 1.7 (Nav1.7). Intrazelluläre Gb3 Ablagerungen wurden unter Verwendung von shRNA-Silencing der α-Galaktosidase A induziert. Nach intraplantarer Injektion von complete Freund‘s Adjuvans (CFA) und chronic constriction injury (CCI) des rechten N. ischiadicus entwickelten alte GLA KO Mäuse, im Gegensatz zu jungen GLA KO und alten WT Mäusen, keine Überempfindlichkeit gegenüber Hitze und mechanischen Reizen. Darüber hinaus fanden wir keine relevanten Unterschiede zwischen Genotypen und Altersgruppen im affektiven und kognitiven Verhalten im naiven Zustand und nach Injektion von CFA. Gen- und Proteinexpressionsanalysen lieferten keine Erklärung für die sensible Beeinträchtigung. Jedoch zeigten kultivierte DRG Neurone von alten GLA KO Mäusen eine deutliche Abnahme der Natriumströme und der Ih Ströme im Vergleich zu jungen GLA KO und alten WT Mäusen. Außerdem wiesen DRG Neurone von alten GLA KO Mäusen eine verstärkte intrazelluläre Akkumulation von Gb3 im Vergleich zu jungen GLA KO und alten WT Mäusen auf. Ähnlich wie bei kultivierten Neuronen waren die Natriumströme in, mit shRNA behandelten HEK-Zellen, im Vergleich zu den Kontrollzellen ebenfalls verringert, konnten aber durch die Behandlung mit Agalsidase-alpha wiederhergestellt werden. Unsere Studie zeigt, dass GLA KO Mäuse ähnlich wie Patienten mit M. Fabry altersabhängige sensible Veränderungen aufweisen. Im Gegensatz zu Patienten sind GLA KO Mäuse jedoch auch vor der Überempfindlichkeit geschützt, die durch eine Entzündung und Nervenläsion hervorgerufen wird. Unsere elektrophysiologischen Ergebnisse jedoch, deuten darauf hin, dass die Reduktion der Natrium- und Ih Ströme mit den veränderten Antworten auf die sensiblen Reize zusammenhängt. Diese Daten lassen auf eine Gb3 abhängige Ionenkanaldysfunktion in DRG Neuronen als potentiellen Faktor für sensible Fehlfunktion und Schmerz bei M. Fabry schließen.

Zugl.: Hannover, Tierärztliche Hochsch., Diss., 2008.

Thesis (Ph. D.)--University of Oregon, 2000. / Title from title screen. Paging within document: xii, 211 p. : ill. (some col.). Includes vita and abstract. Includes bibliographical references (p. 199-211).

Thesis (Ph. D.)--University of Oregon, 2000. / Includes vita and abstract. Includes bibliographical references (p. 199-211). Also available for download via the World Wide Web; free to University of Oregon users.

Submitted by Antonio Geraldo Couto Barreto (ppgms@ufba.br) on 2013-11-04T14:01:42Z No. of bitstreams: 1 PDF Final.pdf: 7736859 bytes, checksum: d029585d5add34bb5b7f5929fdd32069 (MD5) / Approved for entry into archive by Patricia Barroso (pbarroso@ufba.br) on 2013-11-04T17:16:06Z (GMT) No. of bitstreams: 1 PDF Final.pdf: 7736859 bytes, checksum: d029585d5add34bb5b7f5929fdd32069 (MD5) / Made available in DSpace on 2013-11-04T17:16:06Z (GMT). No. of bitstreams: 1 PDF Final.pdf: 7736859 bytes, checksum: d029585d5add34bb5b7f5929fdd32069 (MD5) / INTRODUÇÃO: A doença de Fabry (DF) é um erro inato do metabolismo dos glicoesfingolípidos devido à deficiência da atividade da enzima α-galactosidase A; é ligada ao cromossomo X e pacientes do sexo masculino geralmente apresentam sintomas clássicos. Não há mutações comuns para este gene; mais de 600 mutações já foram descritas no Human Gene Mutation Database. OBJETIVO: O objetivo deste estudo é relatar todas as mutações e alterações polimórficas observadas nos membros de cinco famílias com a doença de Fabry do estado da Bahia-Brasil. MATERIAL E MÉTODOS: Um total de 48 pacientes com suspeita de DF foi encaminhado para avaliação no Serviço de Genética Médica do Hospital Universitário Professor Edgard Santos durante o período de janeiro de 2009 a dezembro de 2011. Os 14 pacientes (06 homens/08 mulheres) com diagnóstico de Doença de Fabry envolvidos neste trabalho são acompanhados no Ambulatório de Erros Inatos do Metabolismo do Hospital Professor Edgard Santos - Universidade Federal da Bahia. Eles pertencem a cinco famílias (I, II, III, IV e V) não relacionadas. O gene GLA foi analisado através da amplificação por PCR (reação em cadeia da polimerase) e sequenciamento de ambas as cadeias de DNA de toda a região codificadora e de regiões nas junções entre éxons e íntrons. Para todos os pacientes do sexo masculino e alguns do sexo feminino foi realizada a medida da atividade da enzima α-galactosidase em gota de sangue seca através da espectrometria de massa em tandem. RESULTADOS: Quatro diferentes mutações causadoras de DF foram encontradas e todas elas já foram relatadas previamente. Duas das cinco famílias compartilham a mesma mutação (p.A156D). Nas famílias I e V (ambas com mutação p.A156D) foi encontrado o mesmo polimorfismo (c.1000-22C> T), mas uma outra diferente variação foi encontrada apenas na Família I. DISCUSSÃO: Apesar do pequeno número de pacientes envolvidos neste estudo, este trabalho é o mais completo em análise de polimorfismos do gene GLA, incluindo todos os pacientes diagnosticados com a doença de Fabry do estado da Bahia-Brasil. A análise de alterações polimórficas sugere que duas das cinco famílias têm uma mesma origem ancestral. Mais estudos para avaliar esta hipótese estão ocorrendo neste momento, através da análise de haplótipos.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico. Programa de Pós-Graduação em Engenharia de Alimentos. / Made available in DSpace on 2012-10-23T11:30:49Z (GMT). No. of bitstreams: 1 255934.pdf: 897286 bytes, checksum: a9c37d8b67e86e7e36ff1b0b486aaa09 (MD5) / A hidrólise enzimática da lactose presente no soro lácteo é uma das possibilidades mais atrativas para utilização deste composto que é produzido em milhões de toneladas anualmente em todo o mundo. Atualmente, parte deste soro é utilizada para nutrição humana e animal e o restante é descartado no meio ambiente. Além da lactose, o soro apresenta em sua composição proteínas de alto valor biológico que podem ser concentradas por evaporação, precipitação ou ultrafiltração. Uma das possibilidades para valorização do soro lácteo com relação à lactose é sua hidrólise em reatores a membrana que apresenta a vantagem de permear continuamente o hidrolisado, mantendo-se a enzima ativa no retentado. Desta forma, maior quantidade de lactose pode ser hidrolisada por unidade de enzima. Sabe-se que enzimas podem interagir com superfícies hidrofóbicas, que são comuns em muitas membranas poliméricas. Portanto, é importante pesquisar se esta interação pode levar a uma perda de atividade catalítica da enzima adsorvida. Isto pode ocorrer devido à possibilidade da enzima utilizar o seu sítio ativo na adsorção, perdendo parte de sua atividade. Este trabalho teve como objetivo estudar a hidrólise da lactose a partir do permeado resultante da concentração de leite por ultrafiltração, verificando-se a influência das peptonas sobre a ação da enzima ß-galactosidase. Verificou-se, também, a atividade da enzima adsorvida na superfície da membrana e a capacidade de retenção da enzima pelas membranas. A hidrólise da lactose foi estudada em reator a membrana de fluxo perpendicular. Foram utilizadas membranas planas de polifluoreto de vinilideno (PVDF) e de poliétersulfona (PES), ambas com ponto de corte de 10 kDa. Foram utilizadas duas marcas comerciais de ß-galactosidase (Kluyveromyces lactis), Lactozym® 3000 L HP-G e Maxilact® L-5000. A taxa de hidrólise foi determinada através da pesquisa de glicose, um dos produtos da hidrólise, através de kit colorimétrico-enzimático. A maior atividade da enzima Lactozym foi na concentração de 0,4 g.L-1, a 40°C e pH 6,8 e a melhor atividade da enzima Maxilact, na concentração de 0,2 g.L-1, a 35°C e pH 6,8. Os resultados mostraram que a membrana foi capaz de reter totalmente a enzima lactase no reator, conseguindo-se até 95% de hidrólise da lactose após 90 minutos de processo, à temperatura de 35oC e pH 6,8. As peptonas do permeado foram removidas através de aquecimento e centrifugação e seu efeito sobre o fluxo permeado e a taxa de hidrólise foi investigado. A remoção das peptonas do soro fez aumentar o fluxo permeado em até 150% e a taxa de hidrólise em até 20%. Ficou evidenciado que a enzima adsorvida na superfície da membrana apresentou pouca atividade hidrolítica.