Dissertations / Theses on the topic 'Β-galactosidases'

In this thesis, the field of the bifidobacteria! physiology in relation to β- and α-galactosidases is described and perspectives of them for oligosaccharides synthesis are discussed- The experimental work focused on the isolation and characterisation of these cell constituents from Bifidobacterium bifidum NCIMB41171. To generate a basis for subsequent work the construction of a genomic DNA library was carried out. This resulted in the isolation of four different β-galactosidases (bbgI, bbgII, bbgIII and bbgIV) and one α-galactosidase (melA) genes. Molecular characterisation of the gene products indicated their putative location, which was subsequently confirmed after expression in an E. coli host. Nucleotide and amino acid sequence comparisons with other known bifidobacterial enzymes revealed aspects of either their evolution or conservation among species.

This thesis describes efforts made towards the synthesis of a biologically stable, luminescent molecular probe, which could be used to investigate in vivo the processing of sugars by β-galactosidases. To this end, a lactose-based probe was designed, featuring a Lanthanide held within a chelate and appended to the glucosyl unit, and a proximal naphthyl moiety, attached to the galactose unit, which would function as a sensitiser for luminescence. A β-galactosidase enzyme from B. circulans was chosen to carry out the investigation. A number of novel methyl glucosides, functionalised with a naphthyl moiety at C6 of the sugar, were prepared. These were then used as glycosyl acceptors to make disaccharides (lactose analogues), with the enzyme (functioning in reverse) catalysing the glycosylation. The enzymatic reaction was optimised by varying the amount of enzyme, the reaction pH, the ratio of glycosyl acceptor to donor, the reaction temperature, concentration and solvent mixture. The optimal conditions were found to be a 0.4 M reaction solution at pH 7.0 with 20% acetonitrile, a 7:1 ratio of glycosyl acceptor to donor, 19.6 U of enzyme per mmol of acceptor, and a reaction temperature of 30°C. The resulting disaccharide products exhibited unusual regioselectivity for the β-galactosidase from B. circulans, with unexpected β(1→3) and β(1→2) glycosidic linkages being formed. In an effort to increase the efficiency of the process of identifying suitable substrates for the enzyme, a dynamic combinatorial chemistry approach was also explored. This used disulfide bonds to attach the naphthyl moiety to the methyl glucoside using linkers of different lengths. From this library, the enzyme successfully processed the novel disulfide GlcOMe-S-S-CH\(_2\)Np as a glycosyl acceptor with p-nitrophenyl galactose as the glycosyl donor. This resulted in a novel disaccharide featuring a naphthyl group attached via a disulfide bond to the glucosidic residue.

L’ingénierie des compléments alimentaires solides offre plusieurs avantages dans la formulation industrielle des produits alimentaires, en termes de production, stockage, et manipulation. Pour ces raisons, l’objectif de cette thèse était d’élaborer des ‘cargos’ bio-réactifs, permettant l’encapsulation d’une enzyme exogène capable de réaliser la réaction d’hydrolyse des molécules de lactose. Aujourd’hui il est établi que les symptômes caractéristiques de l’intolérance au lactose sont associés à une carence en lactase dans le gros intestine. Ainsi, fournir au corps humain de la lactase est l’application ciblée par ce travail, par la conception de matériaux siliciques comme support d’encapsulation. En général, les types de cargos développés doivent surmonter les conditions gastriques pour libérer l’enzyme dans le gros intestine. La silice poreuse amorphe est un matériau inorganique non-toxique qui assure une bonne protection dans des conditions acides et permet une libération contrôlée au pH légèrement basique du colon. L’utilisation de silice amorphe poreuse permet à coût réduit d’obtenir une structure intrinsèque contrôlée (forme, taille particulaire, diamètre du pore) et une chimie de surface modifiable. En accord avec les objectifs principaux, quatre stratégies d’encapsulation bio-adaptées ont été étudiées comme de potentiels voies pour la production de compléments alimentaires solides d’intérêt pour le traitement de l’intolérance au lactose : (i) immobilisation de l’enzyme par adsorption dans des matériaux siliciques meso-macroporeux pré-synthétises, (ii) immobilisation de l’enzyme sur des particules de silice faiblement poreuses recouvertes par des liposomes, (iii) encapsulation de l’enzyme dans des nanoparticules de lipides solides (SLNs), (iv) encapsulation de l’enzyme dans une matrice de biopolymère recouvert d’une coque de silice mésoporeuse
The engineering of solid dietary supplements provides several advantages in the industrial formulation of food products, in terms of its production, storage and handling. Thereby, the goal of this doctoral work is to design bio-responsive carriers for the encapsulation of an exogenous enzyme able to catalyze the hydrolysis of lactose towards simple sugar molecules. In fact, there is a consensus that the onset of symptoms characteristic of lactose intolerance are associated with lactase deficiency in the small intestine. Providing the organism with exogenous lactase is the underlying application targeted by this work through the design of silicabased materials for encapsulation. The different types of bio-carriers developed had to overcome the simulated gastric conditions in order to release active enzyme molecules in the small intestine. Amorphous porous silica is a very good and non-toxic component affording protection versus acidic conditions, while providing controlled release. This inorganic material approved by the US Food and Drug Administration (FDA) has a relatively low cost, and presents a controlled structure (shape, size, pore diameter), as well as tunable surface chemistry. In agreement with the main objectives, four bio-adapted encapsulation strategies were investigated as potential routes to produce solid dietary supplements for lactose intolerance treatment: (i) physical entrapment of the enzyme in pre-synthesized meso-macroporous silica materials, (ii) physical entrapment of the enzyme in low porosity silica particles coated by liposomes, (iii) encapsulation of the enzyme into thermosensitive solid lipid nanoparticles (SLNs) (iv) encapsulation of the enzyme into a biopolymer matrix coated in a mesoporous silica shell

La micro et la nanotechnologie a créé un bouleversement dans beaucoup de domaine tel que l’industrie ou la recherche. Pour la recherche, les enjeux économiques (quantité) et écologiques (déchets, risques chimiques) vont directement dans le sens de cette miniaturisation pour l’obtention de procédé sûr, propre et moins couteux. Cette thèse présente la mise en place d’un nouveau procédé de conception de BioMEMS assistée par plasma froid. L’objectif est le développement d’un microdispositif à partir d’un matériau non toxique, le Tetramethyldisiloxane (TMDSO), grâce à une technologie de dépôt de couche mince assisté par plasma, et intégrant une enzyme, pour la réalisation de réaction catalytique. Pour cela, un protocole d’immobilisation et d’intégration de l’enzyme, la β-galactosidase, a été développé afin de vérifier la capacité du TMDSO à retenir les enzymes et conserver sa fonction biologique. Ensuite, une évaluation de l’activité catalytique de l’enzyme immobilisée a été entreprise par la réalisation de réaction à l’échelle millifluidique, validant l’immobilisation ainsi que la biocompatibilité du ppTMDSO. Ensuite, un microréacteur à enzyme immobilisée a été réalisé, afin d’évaluer l’influence du passage à l’échelle microfluidique et de comprendre les phénomènes liés à la diffusion et la réaction des espèces au sein du dispositif. Enfin, la conception d’un microcanal en ppTMDSO et intégrant l’enzyme, a été réalisée afin de d’étudier la faisabilité d’une méthodologie « bio-integrante » pour la création d’un BioMEMS. L’utilisation d’une méthodologie bio-integrante peut être considérée comme une alternative prometteuse pour le développement de nouveaux outils de recherches
The micro and nanotechnology has created an upheaval in many field such as industry or research. For research, economic issues (quantity) and ecological (waste, chemical hazards) go straight in the direction of this miniaturization process for obtaining safe, clean and less expensive. This thesis presents the development of a new BioMEMS design process assisted by cold plasma. The objective is to develop a micro-device from a non-toxic material, tetramethyldisiloxane (TMDSO), through a plasma enhanced thin film deposition technology, and incorporating an enzyme, for carrying out catalytic reaction. For this, an immobilizer protocol and integration of the enzyme, β-galactosidase, was developed to verify TMDSO's ability to retain enzymes and retain its biological function. Then, an evaluation of the catalytic activity of the immobilized enzyme was carried out by carrying out the reaction millifluidic scale, validating the asset and the biocompatibility of ppTMDSO. Then, an immobilized enzyme microreactor was conducted to assess the influence of the transition to the microfluidic scale and understand the phenomena related to the diffusion and reaction of the species within the device. Finally, the design of a microchannel ppTMDSO and incorporating the enzyme, was conducted to study the feasibility of a "bio-integral 'methodology for establishing a BioMEMS. The use of a bio-integral method may be regarded as a promising alternative for the development of new research tools

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A enzima β – galactosidase é reconhecida por catalisar a hidrólise da lactose e possibilitar a formação de galactooligossacarídeos. O objetivo do presente trabalho foi estudar diferentes condições de imobilização de β – galactosidases de Aspergillus oryzae e Kluyveromyces lactis utilizando suporte comercial Immobead. Ambas as enzimas foram imobilizadas nos suportes tratados e não tratados com etilenodiamina e submetidas a processos de imobilização uni e multipontual. Os derivados também foram avaliados quanto ao bloqueio dos grupamentos epóxi com glicina. As análises de estabilidade ao armazenamento sob refrigeração, estabilidade térmica, ciclos de reuso para hidrólise da lactose, determinação das propriedades cinéticas e determinação de pH e temperatura ótimos foram realizadas nos derivados obtidos. Os resultados de eficiência de imobilização variaram de 30 a 50% e os valores de rendimento variaram entre 80 e 90%. Modificações químicas no suporte foram realizadas utilizando etilenodiamina com o objetivo de gerar modificações químicas no suporte, causando a rápida adsorção de enzimas e favorecendo a formação de ligações covalentes multipontuais em tempo reduzido. Verificou-se que suportes modificados com etilenodiamina imobilizaram a mesma carga de enzimas em menor tempo, quando comparados a suportes sem modificação. Entretanto, a significativa perda de atividade verificada nesses suportes durante os ciclos de reuso sugere que a superfície do suporte possa ter sido modificada em sua totalidade, dificultando a formação de ligações covalentes e permitindo a lixiviação de enzimas para o meio reacional. Derivados não bloqueados apresentaram perda considerável de atividade enzimática durante a armazenagem, indicando a ocorrência de possíveis distorções da enzima, ocasionadas pela interação de grupamentos epóxi livres. Ensaios submetidos à imobilização multipontual apresentaram melhorias em sua estabilidade térmica. Os valores de Km para as enzimas imobilizadas de K. lactis e A. oryzae foram 49,69 e 55,29 mM, valores superiores àqueles verificados para as enzimas livres (19,11 e 17,37 mM, respectivamente), indicando possíveis alterações conformacionais na estrutura da proteína, resultantes do processo de imobilização. Os resultados indicaram que derivados não tratados com etilenodiamina, submetidos à imobilização covalente multipontual e bloqueio com glicina apresentaram os resultados mais expressivos para as condições estudadas de estabilidade ao armazenamento, estabilidade térmica e ciclos de reuso para hidrólise de lactose. Esses derivados não apresentaram distorções em relação às condições ótimas de temperatura e pH quando comparadas com as respectivas enzimas livres. As β – galactosidases de A. oryzae e K. lactis submetidas à imobilização covalente multipontual no suporte Immobead posteriormente bloqueado com glicina apresentaram as melhores propriedades para futura aplicação industrial.

Dissertação(mestrado)-Universidade Federal do Rio Grande, Programa de Pós-Graduação em Engenharia e Ciência de Alimentos, Escola de Química e Alimentos, 2009.
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Esse projeto desenvolveu um processo simultâneo de catálise e fermentação láctica visando obter um iogurte com características nutracêuticas. O objetivo principal foi avaliar a conversão da lactose e a síntese de galactooligossacarídeos (GOS) para um substrato específico, comparando biocatálises conduzidas simultaneamente à fermentação com os processos sem adição de enzima. A fermentação foi realizada a partir de cultura láctica liofilizada comercial contendo dois microrganismos probióticos, Bifidobacterium animalis e Lactobacillus acidophilus, associados aos microrganismos característicos do iogurte, Lactobacillus delbruekii subs. bulgaricus e Streptococcus salivarius subs. thermophilus. Foi utilizado um preparado enzimático contendo β- galactosidases obtidas de duas origens distintas: Kluyveromyces lactis e Aspergillus niger. Foram avaliados os efeitos das variações da concentração de lactose no substrato, da concentração de enzima e do tempo de adição da enzima em um planejamento experimental 23. As respostas foram o tempo de processo, a lactose final, a conversão da lactose, a densidade, a viscosidade, a sinérese e a concentração de GOS, comparando os processos enzimáticos e fermentativos simultâneos com a fermentação sem a adição de enzima. Os resultados indicaram um percentual de conversão da lactose entre 97,7 e 99,7% e uma produção de GOS nas condições de maior concentração inicial de lactose no substrato, menor concentração de enzima e maior tempo de defasagem na adição da enzima. Os efeitos sobre os parâmetros de textura foram negativos, indicando a necessidade de um acréscimo de agentes espessantes e estabilizantes nos bioprocessos simultâneos, quando do aumento da concentração de enzima. Observou-se, ainda, um impacto positivo no tempo de processamento quando da comparação entre os bioprocessos simultâneos e os processos de múltiplos estágios, na elaboração de fermentados lácticos com baixa concentração de lactose.
This project developed a simultaneous process of catalysis and lactic fermentation aiming to obtain a yogurt with nutraceuticals characteristics. The main objective was the conversion of lactose and the synthesis of galactooligosaccharides (GOS) for a specific substrate, comparing the biocatalysis conducted simultaneously to the fermentation with the processes without adding enzymes. Fermentation started with a commercial lactic lyophilized containing two probiotics microorganisms, Bifidobacterium animalis and Lactobacillus acidophilus, associated with microorganisms characteristics of yogurts, Lactobacillus delbruekii subs. bulgaricus and Streptococcus salivarius substhermophilus. It was used an enzymatic preparation containing β-galactosidases obtained from two distinct sources: Kluyveromyces lactis and Aspergillus niger. It were evaluated the effects of the variation of lactose concentration on the substrate, the enzyme concentration and the time of enzyme addition in an experimental design 23. The results were the process time, final lactose, lactose conversion, density, viscosity, sineresys and GOS concentration, comparing simultaneous enzymatic and fermentation processes with fermentation without the addition of enzymes. Results indicated a lactose conversion percentage between 97,7% and 99,7%, and a production of GOS in the condition of higher initial concentration of lactose in the substrate, lower enzyme concentration and more time of delayed in the addition of enzyme. The effects on texture parameters were negative, indicating the need for an increase of thickening agents and stabilizers in simultaneous bioprocesses when the increasing the enzyme concentration. It was also observed a positive impact on processing time when it was compared the simultaneous bioprocesses with the multiple stages processes in the elaboration of lactic fermented with low concentration of lactose.

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.

BEZERRA, C. S. Imobilização de β-galactosidase de Kluyveromyces lactis em diferentes suportes e protocolos de ativação. 2012. 115 f. Dissertação (Mestrado em Engenharia Química) - Centro de Tecnologia, Universidade Federal do Ceará, Fortaleza, 2012.
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β-galactosidase immobilization was studied seeking to add value to cheese whey trough lactose hydrolyze producing galactose and glucose. This work aimed to develop biocatalysts using different organic supports and activation protocols. Firstly, some supports were prepared as chitosan 2.5% (w/v) (with and without pretreatment with dimethylformamide) and 2.0% (w/v), chitosan-alginate-epoxide (QAE), cashew bagasse (BC) and coconut shell fiber (CV), which were activated in different ways with glutaraldehyde, epichlorohydrin or glycidol. Initially, it was determined the immobilization yield, couple yield and apparent activity from obtained catalysts, being chosen six derivatives according to better results parameters: 2.5% chitosan (w/v) glutaraldehyde activated (QUITGLU1), 2.0% chitosan (w/v) KOH coagulated at 50°C glutaraldehyde activated (QUITGLU2) and epichlorohydrin (QUITEPI) or glycidol (QUITGLI), chitosan 2.5% (w/v) dimethylformamide treated with epichlorohydrin (QUIT-DMFEPI) or glycidol (QUIT-DMFGLI). Thus, catalysts (QUITGLU1, QUITGLU2, QUITEPI, QUITGLI) were studied as operational stability by using a continuous reactor, as well as, maximum enzyme loading and effectiveness assays. Then, it was determined QUITGLU2 as the best biocatalyst and following studies were carried out: immobilization time, enzyme optimum temperature and pH, kinetic parameters using lactose as substrate at 37°C, storage at 10°C and operational stability using high load enzyme and cheese whey as substrate. CV and BC supports did not present good results for β-Kluyveromyces lactis galactosidase immobilization, as well as, QAE support. Supports treated with dimethylformamide presented low immobilization yields. The results for QUITGLU2 derivative presented maximum loading of 75 mgProtein.g-1support and higher effectiveness than others. The operational stability for this derivative remained stable, with constant glucose production for 10 h of reaction. Immobilization time of 3h proved enough for the process. The Km and Vmáx values were respectively: free enzyme (46.79 mM and 7,142.86 μmol.(mL.min)-1) and catalyst (69.56 mM and 113.25 μmol.(g.min)-1). During 120 days of storage at 10°C, no decrease derivative hydrolitic activity was noted, demonstrating satisfactory storage stability. Finally, the biocatalyst showed good results as operational stability when used high offered enzyme load (theoretically immobilized load 255.9 mgProtein.g-1chitosan) for cheese whey hydrolysis
A imobilização de β-galactosidase para hidrólise de lactose é uma proposta para agregar valor ao soro de leite com conseqüente produção de galactose e glicose. O objetivo deste trabalho foi desenvolver biocatalisadores a partir de diferentes suportes orgânicos e protocolos de ativação visando à hidrólise de lactose proveniente do soro de leite. Inicialmente, prepararam-se os suportes a serem aplicados no estudo como quitosana 2,5% (m/v) (sem e com pré-tratamento com dimetilformamida) e 2,0% (m/v), quitosana-alginato-epoxilado (QAE), bagaço de caju (BC) e fibra de casca de coco verde (CV), os quais foram ativados de diferentes formas, com glutaraldeído, epicloridrina ou glicidol. Na primeira etapa, determinaram-se o rendimento de imobilização, atividade recuperada e atividade aparente dos diferentes derivados obtidos para assim determinar os seis melhores – quitosana 2,5% (m/v) ativada com glutaraldeído (QUITGLU1), quitosana 2,0% (m/v) coagulada com KOH a 50°C ativada com glutaraldeído (QUITGLU2) ou epicloridrina (QUITEPI) ou glicidol (QUITGLI), quitosana 2,5% (m/v) tratada com dimetilformamida ativada com epicloridrina (QUIT-DMFEPI) ou glicidol (QUIT-DMFGLI). Para segunda fase, os catalisadores (QUITGLU1, QUITGLU2, QUITEPI, QUITGLI) foram estudados quanto à estabilidade operacional com o uso de reator contínuo, assim como ensaios de carga máxima e efetividade. Baseado nestes ensaios determinou-se QUITGLU2 como melhor biocatalisador e realizaram-se os seguintes estudos: variação do tempo de imobilização, determinação da melhor temperatura e pH para atividade enzimática, determinação de parâmetros cinéticos, estocagem sob 10°C e estabilidade operacional com o uso de alta carga enzimática usando soro de leite como substrato. Suportes como CV e BC não apresentaram boa adequação para imobilização de β-galactosidase de Kluyveromyces lactis, assim como o suporte QAE. Suportes com tratamento com dimetilformamida apresentaram baixos rendimentos de imobilização. Os resultados para o derivado QUITGLU2 apresentaram carga máxima de 75 mgProteína.g-1 de suporte e efetividade superiores aos demais. A estabilidade operacional para este derivado apresentou-se estável, visto sua produção de glicose constante por 10 h de reação. O tempo 3 h mostrou-se suficiente para imobilização. Os valores de Km e Vmáx tanto para enzima solúvel (46,79 mM e 7.142,86 μmol.(mL.min)-1) quanto para o derivado (69,56 mM e 113,25 μmol.(g.min)-1). Durante os 120 dias de armazenamento sob 10°C, não houve decréscimo da atividade hidrolítica do derivado, demonstrando ótima estabilidade à estocagem. Por fim, o biocatalisador mostrou bons resultados de estabilidade operacional quando utilizado em alta carga oferecida (255,9 mgProteína.g-1 de quitosana de carga teoricamente imobilizada) para hidrólise de soro de leite

LIMA, M. A. Recuperação e purificação de β-galactosidase de Kluyveromyces lactis utilizando cromatografia de modo misto. 2014. 98 f. Dissertação (Mestrado em Engenharia Química) - Centro de Tecnologia, Universidade Federal do Ceará, Fortaleza, 2014.
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The most important lactases, as far as biotechnological interest is concerned, are those produced by Kluyveromyces yeasts, which are intracellular and currently obtained mostly by submerged-state fermentation. This technique, just as the mainstream biotechnological processes, involves a need for protein and peptide purification from a variety of sources. In this context, one of the most promising notable techniques that can be highlighted is Mixed Mode Chromatography, which allows simultaneous ionic and hydrophobic interactions between the adsorbent and the adsorbate. Thus, the aim of this work was to assess the feasibility of recovery and purification of a Kluyveromyces lactis β-galactosidase, produced via fermentation process, by employing Mixed Mode Chromatography. Unit operations, such as protein precipitation and dialysis were also performed in order to concentrate the enzyme of interest and eliminate cell debris and other interferences inherent in the fermentation medium, something that would result in a decrease in the process yield. The production showed satisfactory results, with mean values for total enzyme concentration of 0.45 mg/mL, enzymatic activity of 77 U/mL and specific activity of 167,9 U/mg. The Purification Factor obtained was 1.17. A precipitation step, followed by a dialysis process, was performed and the later chromatographic run carried out in fixed bed with this material yielded recovery values of 41.0 and 48.2% of total protein and activity, respectively. SDS-PAGE Electrophoresis confirmed the purification evolution throughout the unit operations employed, confirming the viability of the employment of the techniques used to obtain an enzyme of considerable degree of purity and possessing high-added value.
As mais importantes lactases, em termos de interesse biotecnológico, são aquelas produzidas por leveduras do gênero Kluyveromyces, que são intracelulares e, em sua maioria, são obtidas por fermentação em cultura submersa. Esta técnica, assim como a maioria dos processos biotecnológicos, envolve a necessidade de purificação de proteínas e peptídeos a partir de uma variedade de fontes. Neste contexto, uma das técnicas mais notavelmente promissoras é a cromatografia de modo misto, que permite interações iônicas e hidrofóbicas simultaneamente entre o adsorvente e o adsorbato. O objetivo do presente trabalho foi estudar a viabilidade da recuperação e purificação da enzima β-galactosidase, produzida por meio de processo fermentativo e utilizando o micro-organismo Kluyveromyces lactis, por técnica de cromatografia de modo misto. Operações unitárias de precipitação proteica e diálise foram também realizadas com o intuito de concentrar a enzima de interesse e eliminar detritos celulares e outros interferentes advindos do meio de fermentação, o que ocasionaria uma diminuição do rendimento do processo. A produção se apresentou satisfatória, com uma média de valores de concentração de enzimas totais de 0,45 mg/mL, atividade enzimática de 67 U/mL, atividade específica de 167,9 U/mg. O Fator de Purificação obtido foi de 1,17. Uma precipitação seguida de diálise foi realizada e a posterior corrida cromatográfica em leito fixo com esse material rendeu valores de recuperação de 41,0 e 48,2% de proteína total e atividade total, respectivamente. A análise de eletroforese SDS-PAGE confirmou a evolução do processo de purificação no decorrer das operações unitárias, atestando a viabilidade do emprego das técnicas utilizadas para obtenção de enzimas com considerável grau de pureza com alto valor comercial agregado.

Pichia pastoris is a methylotrophic yeast gaining acclamation for its capabili ties in heterologous protein expression. In contrast to other host organisms such as bacteria or mammalian cells, P. pastoris offers many advantages over its counterparts. For example, P. pastoris is cost-effective in that it can grow to high cell densities on simple media. The optional use of a constitutive (GAP) or inducible (A OXI) promoter and the ab ility to perfo1m post-translational protein modifications are additional qualities that make for a powerful heterologous expression system. This study focuses on harnessing the benefits described to develop a high-throughput reporter system for the screening of potential super-secreting mutant strains of P. pastoris. Plasmid constructs were engineered with the lacZ reporter gene, which encodes for the β-galactosidase protein, and fused to the S. cerevisiae MATa signal sequence. Expression plasmids were successfully transformed in P. pastoris strain yGS 115 followed by induction. Western blot analyses confirm the expression of β-galactosidase and colorimetric activity assays further validate enzymatic function. A mutant library containing cis- and/or trans-acting mutations was created by treating P. pas loris clones harboring the β-galactosidase expression plasmid with ultraviolet (UV) radiation. A colorimetric plate assay was combined with a replica-plating system that enabled the screening of thousands of potential super-secreting mutant colonies in a high-throughput format. This study sheds light onto our current understanding of secretion in yeast and further contributes to developing P. pastoris into a valuable heterologous protein expression system.

This research explores various immobilized enzyme support materials, including the novel nylon-6 fiber membrane (NFM), observing the increase of surface area and what effect that has on enzyme binding potential. This study also manipulates incubation and reaction conditions and observes what affect that has on activity and stability of β-galactosidase comparing various solid support materials and free enzyme. Nylon-6 fiber membranes were created using the process of electrospinning and were compared with other materials as solid support materials for enzyme binding. The other materials included polyvinylidene fluoride 5 kD nanofiltration dairy membranes, nylon-6 pellets, silica glass beads, and free—dissolved—enzyme. Scanning electron microscopy images exposed the nylon-6 fiber membrane’s large amount of surface area which coordinated with greater enzyme activity as compared to the relatively flatter surfaces of the other solid support materials. Enzyme activity was measured spectrophotometrically with the color-changing substrate ortho-Nitrophenyl-β-galactoside. NFM had greater maximum enzyme binding potential than the other solid supports. Across pH conditions ranging from 3.5 to 6.0., enzyme activity was maintained on the membrane immobilized samples whereas free enzyme did not maintain activity. Altering storage temperature (4, 22, and 50 °C) affected enzyme stability, the ability of the enzyme to maintain activity over time, of free and polyvinylidene fluoride membrane samples. However, nylon-6 fiber membrane samples maintained stability across the varying storage temperatures. Increasing the immobilization solution enzyme concentration above maximum enzyme binding capacity had no significant effect on enzyme stability for membrane immobilized samples. Although, both had lower mean stability than free enzyme by approximately 74% percent. With further development, β-galactosidase immobilized on nylon-6 fiber membranes, or other membranes, could be used in continuous processing in the dairy industry for a combination of filtration and lactose hydrolysis—creating products reduced in lactose and increased in sweetness with no “added sugars” requirement for a nutrition label and no enzyme listed as final product ingredient.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
A região codificadora do gene LAC4, que codifica a β-galactosidase de Kluyveromyces marxianus var. lactis, foi isolada do plasmídeo pLAC4-12, por meio da técnica de reação de polimerização em cadeia (PCR) com oligonucleotídeos iniciadores, que amplificam um fragmento de aproximadamente 3.000 pb. Com os objetivos de expressar e secretar a β- galactosidase, este fragmento foi clonado em um vetor de expressão e secreção pPIC9 do sistema de expressão de Pichia pastoris. A região codificadora LAC4 intacta e uma versão truncada foram fundidas em fase com a seqüência sinal de secreção do fator-α, sob controle do promotor AOX1. A hidrólise dos DNAs recombinantes obtidos com as enzimas de restrição apropriadas confirmou a clonagem da região codificadora LAC4 intacta nas orientações anti-senso e senso e da versão truncada no vetor pPIC9. Os clones foram linearizados e usados para transformar células eletrocompetentes de Pichia pastoris GS115 (His - e Mut + ). Colônias recombinantes foram isoladas e as inserções da região codificadora LAC4 intacta e truncada no genoma da levedura foram confirmadas por “PCR” com iniciadores específicos. Colônias recombinantes positivas foram cultivadas em glicerol e a expressão dos genes recombinantes foi induzida com metanol. Amostras foram coletadas periodicamente e o meio extracelular e a massa de células analisados quanto à atividade de β -galactosidase. Nenhum aumento na atividade de β - galactosidase foi observado perante o controle. Espera-se que a transformação de linhagens da levedura Mut - com os clones obtidos possa resultar na produção de uma proteína funcional.
The codins Kluyveromyces region marxianus of var. the lactis β-galactosidase was isolated LAC4 from gene, pLAC4-12, from by polimerase chain reaction (PCR) technique with primers that amplify a fragment of approximately 3.000 bp. Expression and secretion vector to expresse and secrete β-galactosidase, the intact and truncated LAC4 coding regions were fused in phase with the factor-α secretion signal sequence in the Pichia pastoris pPIC9. Appropriate restriction enzyme hydrolates confirmed the insertion of the intact LAC4 coding region in sense and antisense orientations, as well as its truncated version in to the pPIC9 vector. The clones were linearized and used to transform electrocompetent Pichia pastoris GS115 (His - e Mut + ) cells. Recombinant cells were isolated and the insertions of intact and truncated LAC4 coding regions in the yeast genome were confirmed by PCR with specific primers. Positive recombinant colonies were grown in glycerol and the expression of the heterologous genes was induced with methanol. Samples were periodically collected and the extracellular medium and cell mass were analyzed for β-galactosidase activity. No increase in β-galactosidase activity was observed over the control. The transformation of Mut - yeast cell lines with the clones is expectial to result in the production of a functional protein.
Dissertação importada do Alexandria

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FACEPE
O objetivo deste trabalho foi imobilizar covalentemente concanavalina-A (Con-A) à nanopartículas magnéticas do tipo magnetita-PANI para purificação da β-galactosidase a partir do fungo Penicillium islandicum URM5073. As condições de imobilização da Con-A e purificação da β-galactosidase, foram aperfeiçoadas através de planejamento fatorial fracionado 2⁶⁻², cuja análise foi realizada em função de duas respostas: quantidade de lectina imobilizada às nanopartículas e β-galactosidase retida na lectina imobilizada, com variáveis independentes a concentração de proteínas, o pH de solução, quantidade de suporte (mg), tempo de reação com o glutaraldeído, concentração do glutaraldeído e tempo de imobilização. A análise estatística mostrou os efeitos estimados das variáveis sobre o processo de imobilização da concanavalina-A em resposta a quantidade de proteína ligada ao suporte. De acordo com esta análise duas variáveis influenciaram significativamente a imobilização de concanavalina-A em suporte magnético, concentração de proteína e pH da reação. A concentração de proteína mostrou efeito positivo significativo, sugerindo que um aumento no valor deste parâmetro aumenta a imobilização de concanavalina-A às nanopartículas. No entanto, pH mostrou efeito negativo significativo, sugerindo que uma redução no valor do parâmetro aumenta a quantidade de proteína fixada ao suporte. A análise de amostras a partir da purificação de β-galactosidase do extrato bruto de Penicillium islandicum, a enzima comercial de Aspergillus oryzae e a β-galactosidase comercial contaminada com caseína e albumina foi realizada usando os dados obtidos a partir da dosagem de proteínas pelo método de dosagem de Lowry e realizada uma eletroforese SDS-PAGE. Esta metodologia indicou que a enzima purificada revelou uma redução de bandas de proteínas, sendo a β-galactosidase indicada com uma massa molecular aproximadamente de 100 kDa. Uma patente será depositada com base nos resultados obtidos da imobilização de Con-A em nanopartículas magnéticas e sua aplicação na purificação de β-galactosidase.
The aim of work was to immobilize concanavalin-A (Con-A) covalently to magnetic nanoparticles of magnetite-PANI type for purification of β-galactosidase produced by the fungus Penicillium islandicum URM5073. The conditions of Com A immobilization and β-galactosidase purification, were optimized by factorial design 2⁶⁻², whose analysis was performed according to two dependent variables: amount of lectin immobilized on nanoparticles and β-galactosidase retained in lectin. The Pareto chart analysis showed the estimated effects of variables on the process of Con-A immobilization in response to amount of protein bound to support. According this analysis, two significantly variables influence the immobilization of concanavalin-A on magnetic nanoparticle, the protein concetration and pH reaction. The protein concentration showed a significant positive effect, suggesting that an increase in the value of this parameter increases the immobilization of concanavalin-A to the nanoparticles. However, pH showed significant adverse effects, suggesting that a reduction in the value of the parameter increases the amount of protein attached to the support. The analysis of samples from the purification of β-galactosidase crude extract fermented by Penicillium islandicum, commercial Aspergillus oryzae enzyme and β-galactosidase contaminated commercial casein and albumin was performed using the data obtained from the protein content Lowry dosage method and used for SDS-PAGE electrophoresis. This method indicated that the purified enzyme showed a reduction of protein bands, with the β-galactosidase indicated with a molecular mass of approximately 100 kDa. A patent shall be paid based on the results of Con-A immobilized on magnetic nanoparticles and its application in β-galactosidase purification.

FREITAS, M. F. M. de. Produção de β-galactosidase por Kluyveromyces lactis NRRL Y1564 em soro de leite e imobilização em quitosana. 2013. 100 f. Dissertação (Mestrado em Engenharia Química) - Centro de Tecnologia, Universidade Federal do Ceará, Fortaleza, 2013.
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In this work, the enzyme β-galactosidase which catalyzes the hydrolysis of lactose to glucose and galactose, was produced by cultivating the micro-organism Kluyveromyces lactis NRRL Y1564 in whey supplemented with yeast extract. The enzyme is an intracellular metabolite, the release enzyme is very important, therefore were studied various chemical and mechanical methods of extraction and stirring with glass beads, sonication, addition of ethanol and toluene, noting also their effects on enzymatic activity. Subsequently, trials were carried out to study the influence of temperature (30, 34, 37 and 40 ° C) in the production of the enzyme from the cheese whey. In the enzyme extraction, using glass beads by a vórtex was more efficient method compared to others evaluated. The enzyme not presented inhibited or denatured when incubated with ethanol, toluene or ethanol-toluene. The optimum temperature for enzyme production by K. lactis NRRL Y1564 was 30 °C with enzymatic activity of 4418.37 U/g of cells at 12 h of fermentation. The enzyme produced was immobilized on chitosan 2.0% w/v. Different activation protocols using glutaraldehyde, epichlorohydrin or glycidol were evaluated. Was also studied, the contact time the enzyme-carrier (3, 5, and 10 hours) to obtain a biocatalyst to produce high yield, recovered activity and half-life in order to hydrolysis of lactose in batch reactor and fixed bed. The influence of temperature and pH on the hydrolysis of lactose was evaluated using as substrate a synthetic solution (lactose 5.0% (w/v) in potassium phosphate buffer 100 mM with 0.1 mM MnCl2) and skimmed milk containing 4.3% w/v lactose. The support shows better results in the parameters of immobilization was chitosan 2% actived with glutaraldehyde and contact time of 5 hours. The biocatalyst produced in this study showed a stability factor of 17.37 and a storage stability of 100% when stored at 4 °C for 90 days. Temperature optimum hydrolysis of lactose was 40 °C and the optimal pH 7.0. The conversion of lactose to 40 °C for this derivative (3.0 U/g) was on average 53% in 10 cycles (consecutive batches). In batch reactor, the conversion to glucose by the hydrolysis of lactose using synthetic solution was approximately 86% for the soluble enzyme (3.8 U/mL) and 83% of the immobilized enzyme (3.8 U/g). The conversion obtained in the hydrolysis of skim milk was 17% for the soluble enzyme and 20% for the immobilized enzyme.
Neste trabalho, a enzima β-galactosidase, que catalisa a hidrólise da lactose em glicose e galactose, foi produzida pelo cultivo do micro-organismo Kluyveromyces lactis NRRL Y1564 em soro de leite suplementado com extrato de levedura. A enzima é um metabólito intracelular, sendo a liberação da enzima para o meio uma condição essencial, por isso estudaram-se diferentes métodos químicos e mecânicos de extração como, agitação com pérolas de vidro, ruptura em ultrassom com pérolas de vidro, adição de etanol e tolueno, observando também, seus efeitos na atividade enzimática. Posteriormente, realizaram-se ensaios para estudar a influência da temperatura (30, 34, 37 e 40 °C) na produção da enzima a partir do soro de queijo. Na extração da enzima, o uso de esferas de vidro em vórtex foi o método mais eficiente quando comparado com os outros avaliados. A enzima, não sofreu inibição ou desnaturação quando incubadas com etanol, tolueno ou etanol-tolueno. A temperatura ótima de produção da enzima por K. lactis NRRL Y1564 foi 30 °C, com atividade enzimática de 4418,37 U/g de células em 12 h de fermentação. A enzima produzida foi imobilizada em quitosana 2,0% m/v. Diferentes protocolos de ativação usando glutaraldeído, epicloridrina ou glicidol foram avaliados. Estudou-se também, o tempo de contato enzima-suporte (3, 5 e 10 horas) a fim de se obter um biocatalisador que apresentasse alto rendimento, atividade recuperada e tempo de meia-vida, visando a hidrólise da lactose em reator batelada e leito fixo. A influência da temperatura e do pH na hidrólise da lactose foi avaliada, usando como substrato uma solução sintética (lactose 5,0% (m/v) e leite desnatado, contendo 4,3% m/v de lactose. O suporte que apresentou melhores resultados nos parâmetros de imobilização foi a quitosana 2% reticulada com glutaraldeído no tempo de imobilização de 5 horas. O biocatalisador produzido nesse estudo apresentou um fator de estabilidade de 17,37 vezes maior que a enzima solúvel, com uma estabilidade de armazenamento de 100% quando armazenada a 4 °C por 90 dias. A temperatura ótima de hidrólise da lactose foi de 40 °C e o pH ótimo foi 7,0 . A conversão da lactose a 40 °C para este derivado (3,0 U/g) foi em média 53% em 10 ciclos (bateladas consecutivas). Em reator batelada, a conversão em glicose a partir da hidrólise da lactose, usando solução sintética, foi aproximadamente 86 % para a enzima solúvel (3,8 U/mL) e 83 % para a enzima imobilizada (3,8 U/g). A conversão obtida na hidrólise do leite desnatado foi de 17 % para a enzima solúvel e 20 % para a enzima imobilizada.

LIMA, Ariosvana Fernandes. Imobilização de uma β-galactosidase produzida por Kluyveromyces lactis NRRL Y-1564 cultivada em soro de leite. 2012. 100 f. : Tese (doutorado) - Universidade Federal do Ceará, Programa de Pós-Graduação em Biotecnologia, RENORBIO, Centro de Ciências, Fortaleza-CE, 2012.
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The enzymatic hydrolysis of lactose by β-galactosidase plays an important role in the processing of dairy products, such as the production of milk containing low concentrations of lactose, the prevention of crystallization in dairy products, and the use of galactosyltransferase for synthesizing galacto-oligosaccharides. In this context, this work aims to study how Kluyveromyces strains can be used to produce β-galactosidase from an agro-industrial by-product such as whey. The species studied were K. marxianus (LAMI CE 025, CCA 510, ATCC 36907) and K. lactis(NRRL Y-1564 and Y-4087). This work also aims to investigate the immobilization of the enzyme onto chitosan and determine its properties such as the optimal operating pH and temperature, the thermal stability of the enzyme, the thermal desnaturation constant, the half-life and the kinetic parameters Km and Vmax using ONPG as substrate of the enzyme β-galactosidase from Kluyveromyces lactis strain NRRL Y1564. K. marxianus LAMI CE 025 and CCA 510 did not consume lactose of the complex medium. The other strains were studied for β-galactosidase production in whey. The maximum enzymatic activity of 3.7 U/mL was achieved by K. lactis NRRL Y-1564 after 12h of fermentation at 180 rpm and 30°C, being selected as a microorganism for β-galactosidase production. The optimal pH for soluble β-gal activity was found to be 6.5 while the optimal pH for immobilized β-gal activity was found to be 7.0, while the optimal operating temperatures were 50°C and 37°C, respectively. The soluble and immobilized enzyme showed similar deactivation profiles at 40°C. For more than 200 min, both biocatalysts showed the same stability, retaining approximately 50 % of their initial activities. However, However, the immobilized enzyme showed an increased stability (8 times) at 50°C. In the lactose hydrolysis at 37°C and pH 7.0 by soluble enzyme was observed a conversion of 58.68% using a enzymatic charge of 2.0 U and 17.57% to 0.5 U. The immobilized enzyme was reused for 10 cycles, showing a good operational stability by retaining more than 74% of its initial activity. The immobilized enzyme retained 100% of its initial activity when it was stored at 4°C and pH 7.0 for a period of 93 days. The soluble β-galactosidase lost 9.4% of its initial activity when it was stored at the same conditions. According to these results, an alternative culture medium prepared by using deproteinized whey supplemented with yeast extract was efficiently used for the production of β-galactosidase through the cultivation of Kluyveromyces strains. Chitosan activated with glutaraldehyde is a suitable alternative low cost support for β-galactosidase immobilization, providing the immobilized enzyme with higher thermal, operational and storage stabilities in comparison with the soluble enzyme.
A hidrólise enzimática da lactose por β-galactosidase desempenha importante papel no processamento de produtos lácteos, sendo uma das aplicações à obtenção de leite com lactose reduzida para o consumo por indivíduos com intolerância à lactose, produção de cápsulas de enzimas para tratamento e a prevenção da cristalização em produtos lácteos. Neste contexto, este trabalho foi desenvolvido visando a seleção de cepas de Kluyveromyces produtoras da enzima β-galactosidase usando um resíduo agroindustrial, o soro de leite como meio de cultivo. Inicialmente, realizou-se a seleção de espécies de Kluyveromyces capazes de produzir β- galactosidase utilizando lactose como fonte de carbono, em meio complexo e posteriormente em soro de leite (50 g/L de lactose) desproteinado e suplementado com extrato de levedura (1 g/L). Após definir a levedura que apresentava maior produção da enzima de interesse, estudou-se a produção e a viabilidade de imobilizá-la em quitosana. Após, caracterizou-se a enzima solúvel e imobilizada, consistindo na determinação do pH e temperatura ótimos, estabilidade térmica, estimativa dos parâmetros termodinâmicos e determinação dos parâmetros cinéticos Km e Vmáx usando como substrato ONPG. As cepas de K. marxianus LAMI CE025 e CCA 510 não consumiram lactose em meio complexo. As demais cepas foram avaliadas quanto à produção de β-galactosidase em soro de leite. A atividade máxima de 3,7 U/mL foi obtida por K. lactis NRRL Y-1564 após 12 h de cultivo a 180 rpm e 30ºC, sendo selecionada como micro-organismo para a produção da β-galactosidase. O pH ótimo para a enzima solúvel e imobilizada foram 6,5 e 7,0, respectivamente, e temperatura ótima de 50 e 37°C para a β-galactosidase solúvel e imobilizada, respectivamente. A enzima solúvel e imobilizada mostrou perfis semelhantes de desactivação a 40 ° C. Durante mais de 200 min, ambos os biocatalisadores mostrou a mesma estabilidade, retendo cerca de 50% da sua actividade inicial. Entretanto, a 50ºC, a enzima imobilizada mostrou uma maior estabilidade térmica, sendo 8 vezes mais estável. Os parâmetros cinéticos Km e Vmáx foram 3,34 mM e 1,78 mM/min para a β-galactosidase solúvel comparado com 3,68 mM e 3,38 mM.min para a enzima imobilizada. Na hidrólise de lactose utilizando a enzima solúvel a 37ºC e pH 7,0 foi verificada uma conversão de 30,77% da lactose para a carga de 2,0 U e 9,8% usando 0,5 U. Após o 10º reciclo de uso, a enzima imobilizada reteve 74% da atividade inicial. A β-galactosidase imobilizada, estocada em tampão fosfato de potássio pH 7,0 a 4°C manteve 100% de sua atividade enzimática inicial no período de 93 dias. A β-galactosidase solúvel perdeu 9,4% de sua atividade inicial quando foi estocada nas mesmas condições. De acordo com os resultados obtidos, o soro de leite mostrou-se uma fonte de carbono alternativa para produção de β-galactosidase de K. lactis NRRL Y1564 e a quitosana ativada com glutaraldeído é um suporte alternativo adequado de baixo custo para imobilização da β-galactosidase, proporcionando a enzima imobilizada estabilidades térmicas, operacional e de armazenamento comparado com a enzima solúvel.

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

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Fundação de Amparo à Pesquisa do Estado de Minas Gerais
A levedura Kluyveromyces marxianus var. lactis (K. lactis) foi cultivada em soro de queijo ultrafiltrado (SUF) em regimes de batelada e contínuo com o objetivo de investigar as condições fisiológicas que levam ao aumento e à queda da atividade de β-galactosidase na entrada da fase de desaceleração do crescimento. As fases fisiológicas do crescimento no cultivo em batelada foram caracterizadas, e observou-se que as concentrações iniciais de células de DO 600 0,1, 0,2 e 0,3 afetam a velocidade de crescimento, porém a delimitação das fases de crescimento é semelhante. A fase estacionária do crescimento foi apenas iniciada em 144 horas, o que permitiu uma longa fase de desaceleração do crescimento. O aumento e a queda na atividade de β-galactosidase foram acompanhados durante os cultivos, assim como a utilização de lactose e a formação e o consumo de etanol, além do perfil eletroforético da β- galactosidase intra e extracelular. Os picos de atividade máxima da enzima foram encontrados no final da fase log e no início da fase de desaceleração nas culturas conduzidas em regime de batelada e no cultivo contínuo na taxa de diluição de 0,09 h^-1. Nessas condições, as concentrações de lactose no meio não se correlacionaram com o máximo de atividade da enzima. Após a queda da atividade máxima, havia ainda lactose no meio e o etanol em concentrações crescentes. Desta forma, a queda na atividade não está relacionada com a exaustão de lactose no meio, nem com o crescimento diáuxico à custa do etanol, embora durante a fase de desaceleração do crescimento tenha sido observada diauxia quando a concentração de lactose era limitante. Outros picos de atividade foram evidenciados antes e após o pico máximo, onde foram obtidos os mesmos resultados. A β-galactosidase das amostras das culturas em batelada e contínua foi analisada em gel de poliacrilamida desnaturante e indicou a inexistência de uma relação direta entre a atividade da enzima e a concentração da proteína, com exceção nos tempos em que a atividade é máxima, quando houve aumento da intensidade da banda protéica no gel. Nas amostras de sobrenadante de ambas as bateladas e da cultura contínua submetidas à análise em gel, não se encontrou β-galactosidase, indicando que o etanol produzido não permeabilizou K. lactis. Cerca de 55 a 69% da lactose em ambos regimes de cultivo, foram convertidos em etanol. E, a variação cíclica da cinética de atividade durante o cultivo em regime de batelada e regime contínuo pode ser explicada pelos eventos de regulação da síntese e da atividade de β-galactosidase.
The yeast Kluyveromyces marxianus var. lactis (K. lactis) was cultivated in ultrafiltered cheese whey (UCW) in batch and continuous culture with the aim to investigate the physiological conditions that lead to the increase and decrease of the activity of β-galactosidase in the beginning of the late log phase of growth. The physiological growth phases were characterized in the batch culture, and it was observed that the initial cell concentration of OD 600 0,1, 0,2 e 0,3 affected the growt h velocity. However, the delimitation of the growth phases is similar. The stationary phase of growth started after 144 hours, which enabled a long late log phase of growth. The increase and the decrease in the β-galactosidase activity were monitored during the cultivation, as well as the use of lactose, the formation and consumption of ethanol as well as the electrophoretical profiles of intra cellular and extra cellular β- galactosidase. The peaks of maximum activity of the enzyme were found in the end of the log phase, and in the beginning of the late log phase in batch culture, and in the continuous culture at the dilution rate of 0,09 h^-1. Under these conditions, the lactose concentrations in the medium did not correlate with the maximum activity of the enzyme. After the maximum activity, there was still lactose in the medium and a rising concentration of ethanol. Therefore, the decrease of activity is not related to the exhaustion of lactose in the medium nor with the diauxic growth due to the ethanol. Albeit, during the late log phase of growth slight diauxic growth was observed when the lactose concentration was limiting. Other peaks of activity were noticed before and after the maximum peak, where the same results were obtained. β- galactosidase of the batch and continuous culture samples was analyzed in denaturing polyacrylamide gel and showed that a direct relationship between the enzyme activity and the protein concentration does not exist, except in the maximum activity times, when there was some increase in intensity in the proteic band of the gel. In the supernatant samples of both batch and continuous culture submitted to gel electrophoresis, β-galactosidase was not found, implying that the ethanol produced did not permeabilize K. lactis. Fifty five to sixty nine percent of the lactose in both cultures was converted to ethanol. And, the cyclical variation in the kinetic of the activity during the cultivation in batch and continuous culture can be explained through the regulation events of the synthesis and the activity.

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.

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Cheese whey is the aqueous portion milk rich in lactose, which separates the curd during cheese manufacture which makes it of great importance effluent dairy industries. With the rapid advances in microbial biotechnology, it is considered good alternative to its use, as a substrate inexpensive and available in obtaining β-galactosidase, which is widely used for pharmaceutical and food industries. The sources most widely used for its production are yeasts, as the species Kluyveromyces lactis and Kluyveromyces marxianus. The objective of this study was to evaluate the feasibility of using the cheese whey, in isolation or in combination with other carbon sources for the production of β-galactosidase enzyme, using Kluyveromyces lactis URM 6684, using the planning simplex centroid mixture. 9 fermentations were carried out using three sources of carbon: cheese whey, lactose and sucrose PA PA. During the study it was determined biomass, pH and after cell disruption, enzymatic activity. With regard to biomass, medium with sucrose had better results E5 (7,8 g / L), E8 (6,8 g / L) and E6 (6,1 g / L). The enzyme activity is presented to best results in medium with cheese whey: E1 (0,65 U / ml), E4 (0,33 U / ml) and E6 (0,40 U / ml). Statistical analysis showed that the enzyme activity showed the best fit to the quadratic and cubic models, which presented the best fit quadratic and cubic models, with coefficients, RA2 = 0.86 and RA2 = 0.93. The results showed that whey was presented as a suitable source of carbon and low cost for production of the enzyme, adding more value to its use, and reduce the costs of production of β-galactosidase enzyme, increasing its availability commerce more affordable and enabling their application in the dairy food industry.
O soro de leite ou de queijo é a porção aquosa do leite, rico em lactose, que se separa do coágulo durante a fabricação de queijos o que o torna efluente de grande relevância das indústrias de laticínios. Com os rápidos avanços em biotecnologia microbiana, considera-se boa alternativa para sua utilização, por ser um substrato barato e disponível, na obtenção de β-galactosidase, que é amplamente utilizada pelas indústrias farmacêutica e de alimentos. As fontes mais utilizadas para sua produção são as leveduras, como as espécies Kluyveromyces lactis e Kluyveromyces marxianus. O objetivo deste trabalho foi avaliar a viabilidade do uso do soro de queijo, de forma isolada ou combinada a outras fontes de carbono, para produção da enzima β-galactosidase, utilizando Kluyveromyces lactis URM 6684, empregando o planejamento de mistura simplex centroide. Foram realizadas 9 fermentações utilizando três fontes de carbono: soro de queijo, lactose PA e sacarose PA. Durante o estudo foram determinados valores de biomassa, pH e, após rompimento celular, atividade enzimática. Com relação à biomassa, meios com presença de sacarose obtiveram melhores resultados: E5(7,8 g/L), E8 (6,8 g/L) e E6 (6,1 g/L). A atividade enzimática apresentou-se com melhores resultados em meios com presença de soro de queijo: E1(0,65 U/mL), E4 (0,33 U/mL) e E6 (0,40 U/mL). A análise estatística evidenciou que a atividade enzimática apresentou melhor ajuste aos modelos quadrático e cúbico, a qual apresentou melhor ajuste aos modelos quadrático e cúbico, com coeficientes, RA2, de 0,86 e 0,93. Os resultados encontrados demonstraram que o soro de queijo apresentou-se como uma fonte de carbono adequada e de baixo custo para produção da enzima, agregando maior valor para seu uso, além de reduzir os custos da produção da enzima β-galactosidase, aumentando sua disponibilidade no comércio a preços mais acessíveis e viabilizando sua aplicação na indústria de alimentos derivados de leite.

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Financiadora de Estudos e Projetos
In this work the immobilization of enzyme β-Galactosidase has been investigated with use of assembly technique of nanostructured films denominated LbL (Layer-by-Layer) for employment in an amperometric biosensor of lactose. Therefore, spectroscopy measured has made for UV-vis (Ultraviolet-visible) and fluorescence, in order to monitor the bands of absorption and emission of each bilayer deposited and to confirm the presence of enzyme in the films. It has made too FTIR (Fourier Transform Infrared Spectroscopy) and SFG (Sum Frequency Generation) for the purpose to understand the films structure, the interactions present and the efficiency of technique in the immobilization process. For the lactose detection amperometric measured have carried and that way the performance of modified electrode have evaluated in relation to enzyme immobilization, the polyelectrolytes performance Poly (allylamine hydrochloride) (PAH) and Poli (etileno imina) (PEI) and poly(ethyleneimine) and the operations conditions of biosensor which sensibility, limit of detection, interferences and stability. The evaluation of enzyme deposition by UV-vis and fluorescence showed that the films growth has been satisfactory presenting the characteristics bands of da β-Gal regarding to amino acid residues tryptophan, tyrosine and phenylalanine, among others, in λ = 280 nm for absorption and λ = 344 nm for emission and the deposition of material has been growing. The spectrum of FTIR and SFG indicated bands of chemical groups characteristics of polymers and enzyme and proved that the bonds, probably secondary of the elements are sufficiently strong for to keep the films in substrate during the sensory evaluation. In lactose detection it has made electrode of (PEI/β-Gal)n e (PAH/β-Gal)n both of ten and third bilayers. The sensibility of film (PEI/β-Gal)10 was 0.061 μA mmol-1 cm-2, while the (PAH/β-Gal)10 was 0.079 μA mmol-1 cm-2. In order to increase the efficiency of the biosensors, electrodes compounds for third bilayers were tested, this way the film (PEI/β-Gal)30 has achieved a sensibility higher than the previous electrodes of 0.31 μA mmol-1 cm-2, is likely that a great amount of enzyme has been immobilized. However, the film ITO/PB/(PAH/β-Gal)30 has not get the same efficiency despite de number of bilayer have been increase. Have been identified two interferences, the glucose and the ascorbic acid, but both can be avoided, the first with use of a biosensor for glucose coupled with lactose biosensor and the second causes an elevation on current, being naturally differentiated. The stability of the biosensor was twelve days, being measured in days alternate. All experiments performed to converge to prove that the LbL technique were adequate to assembly the biosensor and that lactose detection can be done and in levels nearby to real samples, but is possible to improve the system still with studies more expanders about the films structure and to test news biosensors configurations.
Neste trabalho investigou-se a imobilização da enzima β-Galactosidase utilizando a técnica de automontagem de filmes nanoestruturados, denominada LbL (Layer-by-Layer do inglês) para o emprego em um biossensor amperométrico de lactose. Foram feitas medidas espectroscópicas por Ultravioleta e visível (UV-vis) e fluorescência, a fim de monitorar as bandas de absorção e emissão de cada bicamada depositada e confirmar a presença da enzima nos filmes. Fez-se também espectroscopia por transformada de Fourier (FTIR) e Geração de Soma de Frequências (SFG) para compreender a estrutura dos filmes, as interações presentes e a eficiência da técnica no processo de imobilização. Para a detecção de lactose realizou-se medidas amperométricas e dessa forma avaliou-se o desempenho do eletrodo modificado em relação à imobilização da enzima, a eficiência dos polieletrólitos Poli (alilaminahidroclorada) (PAH) e Poli (etileno imina) (PEI) e as condições de operação do biossensor tais como sensibilidade, limite de detecção, interferentes e estabilidade. A avaliação da deposição da enzima pelo UV-vis e fluorescência mostrou que o crescimento dos filmes foi satisfatório, apresentando as bandas características da β-Gal atribuídas aos resíduos de aminoácidos triptofano, tirosina e fenilalanina, entre outros, em λ = 280 nm por absorção e sendo os mesmo responsáveis pela emissão em λ = 344 nm sendo que a deposição de material foi crescente. Os espectros de FTIR e SFG indicaram bandas de grupamentos químicos característicos dos polímeros e da enzima e comprovaram que as interações, provavelmente secundárias, entre os elementos são fortes o suficiente para manter o filme no substrato durante as avaliações sensoriais. Na detecção de lactose foram feitos eletrodos de ITO/PB/(PEI/PVS)1/(PEI/β-Gal)n e ITO/PB/(PAH/PVS)1/(PAH/β-Gal)n ambos com dez e trinta bicamadas. A sensibilidade do filme ITO/PB/(PEI/PVS)1/(PEI/β-Gal)10 foi 0,061 μA.mmol-1.cm-2, enquanto o ITO/PB/(PAH/PVS)1/(PAH/β-Gal)10 foi 0,079 μA.mmol-1.cm-2. A fim de aumentar a eficiência do biossensor eletrodos compostos por 30 bicamadas foram testados, dessa forma o filme ITO/PB/(PEI/PVS)1/(PEI/β-Gal)30 alcançou uma sensibilidade superior aos eletrodos anteriores de 0,31 μA.mmol-1.cm-2, pois é possível que uma maior quantidade de enzimas tenha sido imobilizada. Entretanto o filme ITO/PB/(PAH/PVS)1/ (PAH/β-Gal)30 não obteve a mesma eficiência mesmo tendo o número de bicamadas aumentado, o que pode ser atribuído à estrutura do polieletrólito. Foram identificados dois interferentes, a glicose e o ácido ascórbico, mas ambos podem ser evitados, o primeiro com o uso de um biossensor para glicose acoplado ao da lactose e o segundo causa uma elevação na corrente, sendo naturalmente diferenciado. A estabilidade do biossensor foi de 12 dias, com medida em dias alternados. Todos os experimentos realizados convergem para provar que a técnica LbL foi adequada para a construção do biossensor e que a detecção da lactose pode ser feita em níveis de concentração próximos à de amostras reais, mas ainda é possível aprimorar o sistema com estudos mais aprofundados sobre a estrutura dos filmes e testar novas configurações de biossensores.

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A β-D-galactopiranosídeo hidrolase é uma enzima com ampla aplicação tecnológica, clínica e experimental. A enzima possui um íon de metal bivalente coordenado. Para se obter informações a respeito do centro metálico da enzima, algumas espécies de fungos filamentosos foram testadas para a produção de β−galactosidase, ente as quais, a linhagem de Trichoderma reesei FTKO foi selecionada. O extrato enzimático, ao passar pelo processo de purificação, revelou ao menos duas isoformas de β- galactosidase identificadas como BGT I e BGT II. As enzimas são termofílicas e termotolerantes. A dependência do pH para a atividade, os valores de medidas cinéticas e a atividade revelada em gel de poliacrilamida distinguem as isozimas. Para avaliar a necessidade de metais bivalentes foi padronizada uma metodologia de desativação da enzima, apoiado na competição entre o agente seqüestrante e a enzima pelos metais. A metodologia foi aplicada ao o extrato comercial Lactozym 3000 (Novozyme Latin America Ltd.) revelou a necessidade de metal para a sua atividade com a desativação seguida de reativação pelos íons Mg2+, Mn2+, Co 2+, Ni2+, mas não por Zn2+ e Eu3+. A enzima BGT I (pH ótimo de 4,5) mostrou-se, contudo, resistente à desativação, pois se mantém ativa mesmo em altas concentrações de EDTA.
The β-D-galactopiranosyde-hidrolase is an enzyme with large technological, clinic, and scientific application. The enzyme coordinates a bivalent metal ion. In order to investigate the metal center of the enzyme of moulds, some strains were tested for β−galactosidase production. Among then, Trichoderma reesei FTKO were selected. The partially purified enzymatic extract reveals the production of two isoforms of β-galactosidase, at least. This enzymes are identified as BGT I e BGT II. Both are termophylic and thermotolerant. The pH dependence, the measured kinetic values and the activity revealed in polyacrylamide electrophoresis gels distinguished the isozymes. The metal assistance of enzymatic activity was evaluated in a standard deactivation methodology of enzyme by competition for the metal by chelating agent and metal site of enzyme. When this methodology was applied in the deactivation of commercial extract Lactozym 3000 (Novozyme Latin America Ltd.), revealed the metal importance for activity. The deactivation was reverted by metals ions as Mg2+, Mn2+, Co2+, Ni2+, but not by Zn2+ or Eu3+. On the other hand BGT I enzyme (optimal pH of 4,5) maintained the activity, even in high concentration of EDTA.

The ultimate objective of this work was to develop a real-time method for detecting and monitoring β-D-galactosidase as a suitable indicator of the potential presence of total coliform bacteria in water environments. Preliminary comparison of the chromogenic substrate, chlorophenol red β-D-galactopyranoside and the fluorogenic substrate, MuGAL, revealed unreliable results with the fluorogenic technique due to interference from compounds commonly found in environmental water samples. Thus, the chromogenic assay was further explored. Hydrolysis of the chromogenic substrate chlorophenol red β-D-galactopyranoside by β-D-galactosidase to yield chlorophenol red was the basis of this assay. Fundamental studies with chlorophenol red β-Dgalactopyranoside showed that β-D-galactosidase occurs extracellularly and in low concentrations in the polluted water environment. A direct correlation between enzyme activity and an increase in environmental water sample volume, as well as enzyme activity with total coliform colony forming unit counts were observed. Spectrophotometric detection was achieved within a maximum period of 24 h with a limit of detection level of 1 colony forming unit 100 ml[superscript -1]. This enzyme also exhibited physical and kinetic properties different from those of the pure commercially available β-D-galactosidase. Cell permeabilisation was not required for releasing enzymes into the extracellular environment. PEG 20 000 offered the best option for concentrating β-D-galactosidase. The source of β-D-galactosidase in the polluted environmental water samples was confirmed as Escherichia coli through SDS-PAGE, tryptic mapping and MALDI-TOF, thus justifying the further use of this method for detecting and/or monitoring total coliforms. Several compounds and metal ions commonly found in environmental water samples (as well as those used in water treatment processes) did have an effect on β-D-galactosidase. All the divalent cations except Mg [superscript 2+], at the concentrations studied, inhibited the relative activity of β-D-galactosidase in both commercial β-D-galactosidase and environmental samples. Immobilisation of chlorophenol red β-D-galactopyranoside onto a solid support material for the development of a strip bioprobe was unsuccessful, even though the nylon support material yielded some positive results. A monthly (seasonal) variation in β-Dgalactosidase activity from the environmental water samples was observed, with the highest activity coinciding with the highest monthly temperatures. Electro-oxidative detection and/or monitoring of chlorophenol red was possible. Chlorophenol red detection was linear over a wide range of concentrations (0.001-0.01 μg ml[superscript -1]). Interference by chlorophenol red β-D-galactopyranoside in the reduction window affected analysis. A range of phthalocyanine metal complexes were studied in an attempt to reduce fouling and/or increase the sensitivity of the biosensor. The selected phthalocyanine metal complexes were generally sensitive to changes in pH with a reduction in sensitivity from acidic pH to alkaline pH. The tetrasulphonated phthalocyanine metal complex of copper was, however, more stable with a minimum change of sensitivity. The phthalocyanine metal complexes were generally stable to changes in temperature. While only two consecutive scans were possible with the unmodified glassy carbon electrode, 77 consecutive scans were performed successfully with the CuPc-modified glassy carbon electrode. Among the phthalocyanine metal complexes studied, the CuPc-modified glassy carbon electrode therefore provided excellent results for the development of a biosensor. The CuPc modified-glassy carbon electrode detected 1 colony forming unit 100 ml[superscript -1] in 15 minutes, while the plain unmodified glassy carbon electrode required 6 hours to detect the equivalent number of colony forming units. CoPc, ZnPc and CuTSPc required 2, 2.25 and 1.75 h, respectively, to detect the same numbers of colony forming units. The CuPcmodified glassy carbon electrode detected 40 colony forming units 100 ml[superscript -1] instantly. In general, a direct correlation between colony forming units and current generated in the sensor was observed (R2=0.92). A higher correlation coefficient of 0.99 for 0-30 coliform colony forming units 100 ml[superscript -1] was determined. Current was detected in some water samples which did not show any colony forming units on the media, probably due to the phenomenon of viable but non-culturable bacteria, which is the major disadvantage encountered in the use of media for detecting indicator microorganisms. This novel biosensor therefore presents a very robust and sensitive technique for the detection and/or monitoring of coliform bacterial activity in water.

Resumo: A β-D-galactopiranosídeo hidrolase é uma enzima com ampla aplicação tecnológica, clínica e experimental. A enzima possui um íon de metal bivalente coordenado. Para se obter informações a respeito do centro metálico da enzima, algumas espécies de fungos filamentosos foram testadas para a produção de β−galactosidase, ente as quais, a linhagem de Trichoderma reesei FTKO foi selecionada. O extrato enzimático, ao passar pelo processo de purificação, revelou ao menos duas isoformas de β- galactosidase identificadas como BGT I e BGT II. As enzimas são termofílicas e termotolerantes. A dependência do pH para a atividade, os valores de medidas cinéticas e a atividade revelada em gel de poliacrilamida distinguem as isozimas. Para avaliar a necessidade de metais bivalentes foi padronizada uma metodologia de desativação da enzima, apoiado na competição entre o agente seqüestrante e a enzima pelos metais. A metodologia foi aplicada ao o extrato comercial Lactozym 3000 (Novozyme Latin America Ltd.) revelou a necessidade de metal para a sua atividade com a desativação seguida de reativação pelos íons Mg2+, Mn2+, Co 2+, Ni2+, mas não por Zn2+ e Eu3+. A enzima BGT I (pH ótimo de 4,5) mostrou-se, contudo, resistente à desativação, pois se mantém ativa mesmo em altas concentrações de EDTA.
Abstract: The β-D-galactopiranosyde-hidrolase is an enzyme with large technological, clinic, and scientific application. The enzyme coordinates a bivalent metal ion. In order to investigate the metal center of the enzyme of moulds, some strains were tested for β−galactosidase production. Among then, Trichoderma reesei FTKO were selected. The partially purified enzymatic extract reveals the production of two isoforms of β-galactosidase, at least. This enzymes are identified as BGT I e BGT II. Both are termophylic and thermotolerant. The pH dependence, the measured kinetic values and the activity revealed in polyacrylamide electrophoresis gels distinguished the isozymes. The metal assistance of enzymatic activity was evaluated in a standard deactivation methodology of enzyme by competition for the metal by chelating agent and metal site of enzyme. When this methodology was applied in the deactivation of commercial extract Lactozym 3000 (Novozyme Latin America Ltd.), revealed the metal importance for activity. The deactivation was reverted by metals ions as Mg2+, Mn2+, Co2+, Ni2+, but not by Zn2+ or Eu3+. On the other hand BGT I enzyme (optimal pH of 4,5) maintained the activity, even in high concentration of EDTA.
Orientador: Antonio Carlos Massabni.
Coorientador: Rubens Monti.
Doutor

Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Engenharia e Ciência de Alimentos, Escola de Química e Alimentos, 2008.
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Os galactooligossacarídeos (GOS) vêm sendo considerados como novos ingredientes funcionais dos alimentos, apresentando um grande potencial para melhorar a qualidade dos mesmos, sendo denominados como prebióticos, devido às suas características fisiológicas e tecnológicas. Podem ser produzidos a partir de lactose, principal açúcar presente no soro de leite, através de uma reação enzimática com o uso da enzima β-galactosidase, que possui atividade de transgalactosilação. Este trabalho tem como objetivo principal estudar a obtenção de galactooligosacarídeos por via enzimática. Foi utilizada b-galactosidase de Kluyveromyces lactis Lactozym® 3000L (Novozymes) e, como substratos, a lactose e o soro de leite. Um planejamento experimental 23 totalizando 11 ensaios foi proposto para cada um dos substratos, verificando-se a influência da temperatura (30 a 40°C), concentração de lactose (20 a 40%) e concentração da enzima (5 a 10 U.mL-1), obtendo-se como respostas a concentração de GOS máxima, o rendimento de GOS máximo, a produtividade de GOS máxima e conversão de lactose máxima. Os ensaios foram conduzidos a 180 rpm de agitação, em meio aquoso e pH 7,0 (tampão fosfato de sódio 0,1M), retirandose alíquotas ao longo do tempo, sendo os açúcares presentes em cada amostra quantificados através de um sistema de cromatografia líquida de alta eficiência HPLCPAD (Dionex). A enzima Lactozym® 3000L apresentou atividade de transgalactosilação na presença dos dois substratos. Utilizando como substrato lactose o rendimento atingiu 43,8% e a concentração de GOS foi de 175,3 g/L no sistema reacional composto por 40% de lactose e 10 U.mL-1 de enzima a 30°C. No entanto a produtividade máxima alcançada, de 41,1 g.L-1.h-1, bem como a máxima conversão de lactose (80,5%), foram obtidas em condições de síntese distintas. Utilizando como substrato soro de leite, os valores máximos obtidos para as respostas concentração de GOS (119,8 g.L-1) e rendimento (29,95%) em 4 h de processo foram obtidos no sistema reacional composto por 40% de lactose e 10 U.mL-1 de enzima a 40°C. Nestas condições a produtividade máxima alcançada foi de 64,4 g.L-1.h-1. O máximo valor alcançado para conversão de lactose (87,8%) foi obtido nas mesmas condições de temperatura e concentração de enzima, mas com 20% de lactose.
Galactooligosaccharides (GOS) have been considered as new functional food ingredients, presenting a great potential to improve its quality. They are denominated as prebiotics, due to their physiological and technological characteristics. They can be produced from lactose, the main sugar of the whey, through an enzymatic reaction with the use of the enzyme β-galactosidase, with transgalactosylation activity. The main goal of this work was to study the enzymatic production of galactooligosaccharides. It was used β-galactosidase from Kluyveromyces lactis Lactozym® 3000L (Novozymes) and, as substrates, lactose and whey. A 23 experimental design (11 assays) was proposed for each substrate, verifying the effect of the temperature (30 to 40°C), lactose concentration (20 to 40%) and concentration of the enzyme (5 to 10 U.mL-1). The responses were maximum GOS concentration, maximum GOS yield, maximum productivity and maximum lactose conversion. Assays were carried out at 180 rpm in aqueous medium and pH 7.0 (0.1 M sodium phosphate buffer). Samples were collected and sugars were quantified by high performance liquid chromatography (HPLC-PAD system, Dionex). For lactose the yield reached 43.8% and GOS concentration was 175.3 g.L-1, in the system composed by 40% of lactose and 10 U.mL-1 of enzyme at 30°C. However, maximum productivity (41.1 g.L-1.h-1) as well as maximum lactose conversion (80.5%) were obtained in different conditions. For whey, the maximum values for the responses GOS concentration (119.8 g.L-1) and yield (29.95%) in 4 h of process were obtained in the system composed by 40% of lactose and 10 U.mL-1 of enzyme at 40°C. In these conditions, maximum productivity was 64.4 g.L-1.h-1. The maximum value for lactose conversion (87.8%) was obtained in the same temperature and enzyme concentration, but with 20% of lactose.

Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Engenharia e Ciência de Alimentos, Escola de Química e Alimentos, 2010.
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Este trabalho teve como objetivo principal investigar a imobilização da enzima β- galactosidase comercial de Kluyveromyces lactis (Lactozym®), bem como caracterizar a enzima livre e imobilizada visando sua aplicação na reação de hidrólise da lactose. Foram feitos testes preliminares com três suportes diferentes para a imobilização (alginato, quitosana e Eupergit® C), sendo que os valores de recuperação da atividade enzimática foram, respectivamente, 2,43%, 6,27% e 41,86%, selecionando-se o suporte Eupergit® C como o mais promissor. Um planejamento Plackett-Burman, correspondente a 12 ensaios mais 3 réplicas no ponto central, foi proposto a fim de verificar os efeitos das variáveis temperatura, pH, força iônica, tempo de imobilização, concentração de galactose, concentração de íons Mg2+ e volume de enzima na imobilização de β-galactosidase em Eupergit® C. A força iônica e o pH foram as variáveis que apresentaram efeito significativo (p<0,1) na imobilização, sendo que o aumento da força iônica de 0,1 M para 1,5 M e o aumento do pH de 6,6 para 7,4 representaram aumento de 30,0% e redução de 17,3% na recuperação da atividade enzimática, respectivamente. À temperatura de 25°C, pH 6,6, concentração salina de 1,5 M, tempo de imobilização de 8 h, concentração de Mg2+ de 1 mM e 0,4 mL de enzima adicionada, atingiu-se 85% de recuperação da atividade enzimática. As enzimas livre e imobilizada, nas melhores condições em Eupergit® C, foram caracterizadas quanto aos perfis de pH e temperatura, estabilidade térmica, parâmetros cinéticos e termodinâmicos. Quanto aos perfis de pH e temperatura, foram obtidos, para ambas, valores máximos de atividade enzimática em pH 6,6 e 45°C. Houve um ganho de estabilidade térmica com a imobilização enzimática, tendo-se observado um aumento de cerca de 4 vezes no tempo de meia-vida da enzima imobilizada a 45oC, em relação à livre, o que pode representar vantagens na utilização da enzima imobilizada em escala comercial. Os valores de energia de ativação para as enzimas livre e imobilizada foram, respectivamente, iguais a 35,28 kJ.mol-1 e 29,46kJ.mol-1; e os valores de energia de ativação da reação de desnaturação para as enzimas livre e imobilizada foram iguais, respectivamente, a 266,12 kJ.mol-1 e 198,77 kJ.mol-1. Foram determinados os parâmetros cinéticos para as enzimas livre e imobilizada, sendo que os valores de Km, 30,33 mM e 104,00 mM, respectivamente, representaram uma diminuição da afinidade da enzima pelo substrato com a imobilização, possivelmente devido a fatores estéricos e conformacionais. Parâmetros termodinâmicos foram calculados, evidenciando novamente a maior estabilidade térmica da enzima imobilizada e indicando que a perda de estabilidade de ambas as enzimas, livre e imobilizada, parece não estar associada a alterações relevantes na estrutura terciária.
Immobilization of b-galactosidase (Lactozym®) on Eupergit® C and its characterization This study aimed to investigate the immobilization of commercial β-galactosidase from Kluyveromyces lactis (Lactozym®) and characterize the free and immobilized enzymes for their application in lactose hydrolysis. Three preliminaries tests were carried out with three different supports for immobilization (alginate, chitosan and Eupergit® C), and the values for recovery of enzyme activity were, respectively, 2.43%, 6.27% e 41.86%, selecting Eupergit® C as the most promising. A Plackett-Burman design, composed of 12 assays over 3 central points was proposed in order to verify the effects of temperature, pH, ionic strength, reaction time, galactose concentration, Mg2+ concentration and enzyme volume on β-galactosidase immobilization using Eupergit® C support. The ionic strength and pH were the variables that presented significant effect (p<0.1) on immobilization. The increase in the ionic strength from 0.1 to 1.5 M and the decrease in pH from 6.6 to 7.4 represented an increase of 30.0% and a reduction of 17.3% in the recovery of enzyme activity, respectively. At 25°C, pH 6.6, salt concentration of 1.5 M, immobilization for 8 h, 1 mM Mg2+ and 0.4 mL of enzyme added, reached 85% recovery of enzymatic activity. The free and immobilized enzyme on Eupergit® C were characterized,determining pH and temperature profiles, thermal stability, kinetic and thermodynamic parameters. pH and temperature profiles showed maximum activity at pH 6.6 and 45°C. There was a gain in thermal stability with the enzyme immobilization and there was an increase of about 4 times in the half-life of immobilized enzyme at 45°C, which may represent advantages when using in a commercial scale. The values of activation energy for free and immobilized enzymes were, respectively, equal to 35.28 kJ.mol-1 and 29.46 kJ.mol-1, and the values of activation energy of denaturation reaction for free and immobilized enzymes were equal, respectively, to 266.12kJ.mol-1 and 198.77 kJ.mol-1. Kinetic parameters were determined for the immobilized and free enzyme. Km values of 30.33 mM and 104.00 mM, respectively, represented a decrease of the affinity of the enzyme by the substrate with immobilization, possibly due to steric and conformational factors. Thermodynamic parameters were calculated, showing the higher thermal stability of the immobilized enzyme and indicating that the loss of stability of both enzymes, immobilized and free, does not seem to be associated with significant changes in tertiary structure.

La première partie de notre travail porte sur la réactivation, par traitement à l'urée, de la 13-galactosidase de l'extrait brut d' Escherichia coli inactivée par la température avant et après immobilisation. L'inactivation par la température entraine une perte d'activité de la β-galactosidase soluble et après passage dans l'urée 8M et dialyse de 18 heures avec du tampon, l'activité est régénérée à 75% par rapport à l'ex trait brut initial. La β -galactosidase immobilisée en présence de gélatine par le glutaraldéhyde (0,25%) et inactivée par la température peut être réactivée dans une colonne après traitement à l'urée et lavage du polymère avec du tampon. La cinétique de réactivation de la β-galactosidase immobilisée est complète au bout de 100 minutes environ. Le rendement de l'immobilisation est constant à 0,25 - 0,50 et 0,75% de glutaraldéhyde par contre le pourcentage de réactivation diminue quand la concentration de l'agent pontant augmente. La deuxième partie consiste à mettre au point une électrode enzymatique pour le dosage du L-malate. La flavoprotéine spécifique de l'oxydation du L-malate, élément de la chaîne respiratoire d’ Escherichia coli est induite par la culture des bactéries en milieu minimum en présence de D-L-malate 1% comme seule source de carbone. Les bactéries entières D-L-malate sont immobilisées sur film par inc lusion dans de la gélatine et coréticulation avec du glutaraldéhyde. Les conditions optimales d'immobilisation sont établies, elles concernent la quantité de matériel biologique et les concentrations de gélatine et de glutaraldéhyde. Après immobilisation, la chaîne respiratoire est active et est capable d'oxyder le L-malate en absence de cofacteurs (NAD+, NADP. . . ). Le film enzymatique est mis en contact étroit avec la partie sensible d'une électrode à oxygène (type électrode de Clark). L'addition du L-malate provoque une diminution de la concentration d'oxygène dans le film et donne un signal qui est enregistré. Après avoir analysé le signal émis par l'électrode à L-malate, les constantes cinétiques (VM, K50) ont été déterminées, une courbe de pH a été effectuée, le pH optimum est situé entre 6 et 7, à pH 4 l'activité du film enzymatique n'est réduite que de 50% et permet l'utilisation de l'électrode à L-malate directement dans les milieux biologiques (exemple le vin). L'oxaloacétate (produit de la réaction) et le D-malate inhibent de façon réversible la respiration du L-malate. Le malate peut être dosé dans la gamme de concentration de 0,1mM à 30mM.

Current understanding of potential toxicity of engineered nanomaterials to aquatic microorganisms is limited for risk assessment and management. Here we evaluate if the MetPLATE™ test can be used as an effective and rapid screening tool to test for potential aquatic toxicity of various metal-based nanoparticles (NPs). The MetPLATE bioassay is a heavy metal sensitive test based on β-galactosidase activity in Escherichia coli. Five different types of metal-based NPs were screened for toxicity: (1) citrate coated nAg (Citrate-nanosilver), (2) polyvinylpyrrolidone coated nAg (PVP-nAg), (3) uncoated nZnO, (4) uncoated nTiO2 and (5) 1-Octadecylamine coated CdSe Quantum Dots (CdSe QDs); and compared with their corresponding ionic salt toxicity. Citrate-nAg was further fractionated into clean Citrate-nAg, unclean Citrate-nAg and permeate using a tangential flow filtration (TFF) system to eliminate residual ions and impurities from the stock Citrate-nAg suspension and also to differentiate between ionic- versus nano-specific toxicity. Our results showed that nAg, nZnO and CdSe QDs were less toxic than their corresponding ionic salts tested, while nano- or ionic form of TiO2 was not toxic as high as 2.5 g L− 1 to the MetPLATE™ bacteria. Although coating-dependent toxicity was noticeable between two types of Ag NPs evaluated, particle size and surface charge were not adequate to explain the observed toxicity; hence, the toxicity appeared to be material-specific. Overall, the toxicity followed the trend: CdCl2 > AgNO3 > PVP-nAg > unclean Citrate-nAg > clean Citrate-nAg > ZnSO4 > nZnO > CdSe QDs > nTiO2/TiO2. These results indicate that an evaluation of β-galactosidase inhibition in MetPLATE™ E. coli can be an important consideration for rapid screening of metal-based NP toxicity, and should facilitate ecological risk assessment of these emerging contaminants.

Conselho Nacional de Desenvolvimento Científico e Tecnológico
The study of the enzymatic hydrolysis of the lactose has been of great importance, due to absence or lack of the enzyme present lactase in the process digestive human. The enzyme β-galactosidase is very used in the processing of milk products by being of low cost and enough studied all over the world. The use of this enzyme in the dairy products prevents the crystallization of the products and it provides to the intolerant people the lactose better life quality. The objective of this work was accomplish a comparative study of the hydrolysis of the cheese whey usig two different maufactures β-galactosidase Prozyn and Lactozym, respectively, Kluyveromyces lactis and Kluyveromyces fragilis, of origin of yeasts. The tests of hydrolysis of the cheese whey were accomplished in a reactor with agitation control and temperature. The cheese whey concentration was of 60 g/L, temperatures of 43, 45 and 48°C, concentration of the enzyme of 4 g/L, pH 6,0 and 6,5 and agitation 300 rpm. Both enzymes obteined better result to 45 °C, however the enzyme Prozyn converts the lactose in smaller time. An analysis was accomplished to verify the concentration of total sugar of the cheese whey, 75%, to evaluate the hydrolysis, obtaining the glucose concentration in function of the time for each studied condition. The influence of the lactose concentration was verified in the enzyne, using lactose solution in erlenmeyer. The enzymatic activity was growing of the concentration 10 g/L up to 120 g/. The enzyatic stability in relation to the pH presented better result pH 6,5 for both enzymes Prozyn and Lactozym. The thermal stability of the free enzyme was certain for the time of half life. The enzyme Prozyn to temperature of 50°C it presented time of life 4,62 min and to 55°C was 2,77 min and enzyme Lactozym to the 50°C was 6,32 min and to 45°C was 115,52 min. In the determination of the enzymatic activity the method of the initial rates of the reaction of hydrolysis of the cheese whey was used. The obteined enzymatic activity was 75,03.10-3 mol/(genzyme.h) and 44,6.10-3 mol/(genzyme.h) for Prozyn and Lactozym, respectively.
O estudo da hidrólise enzimática da lactose tem sido de grande importância, devido a ausência ou carência da enzima lactase presente no processo digestivo humano. A enzima β-galactosidase é a mais utilizada no processamento de produtos lácteos por ser de baixo custo, eficiente e bastante estudada em todo o mundo. A utilização desta enzima nos laticínios previne a cristalização da lactose dos produtos e proporciona às pessoas intolerantes a lactose melhor qualidade de vida. O objetivo deste trabalho foi realizar um estudo comparativo da hidrólise da lactose presente no soro de queijo, utilizando β- galactosidase originária das leveduras Kluyveromyces lactis e Kluyveromyces fragilis adquiridas da Prozyn e Lactozym. Os ensaios de hidrólise do soro de queijo foram realizados em um reator com controle de agitação e temperatura. A concentração de soro foi de 60 g/L, temperaturas de 43, 45, 48 e 53°C, concentração da enzima de 4 g/L, pH 6,0 e 6,5 e agitação 300 e sem agitação mecânica. Ambas as enzimas forneceram melhor resultado à 45 °C, no entanto, a enzima Prozyn converte a lactose em menor tempo. O teor de lactose presente no soro de queijo com concentração 60 g/L foi de 75%. A concentração de glicose em função do tempo determinou a duração da hidrólise, onde a lactose foi quebrada pela enzima e resultou na formação de glicose e galactose em quantidade equimolar. Verificou-se a influência da concentração de lactose na enzima, utilizando solução de lactose na faixa de 10 a 120 g/L em solução tampão láctico pH 6,5 e temperatura 30°C. A atividade enzimática foi crescente da concentração 20 g/L até 100 g/L. A estabilidade enzimática em relação ao pH apresentou melhor resultado em pH 6,0 para a Prozyn e pH 6,5 para a Lactozym. A estabilidade térmica da enzima livre foi determinada pelo tempo de meia vida. A enzima Prozyn à 50°C, apresentou tempo de meia vida de 4,62 min e à 55°C, 2,77 min e a enzima Lactozym à 50°C, 6,30 min e à 45°C, 115,52 min. Na determinação da atividade enzimática utilizou-se o método das taxas iniciais da reação de hidrólise do soro. A atividade enzimática obtida foi 75,03.10-3 mol/(genz..h) e 44,6.10-3 mol/(genz..h) para Prozyn e Lactozym, respectivamente.
Mestre em Engenharia Química

Valorizacija permeata kao sporednog proizvoda industrije mleka je od izuzetnog ekološkog, ekonomskog i tehnološkog značaja.Cilj doktorske disertacije je razvoj tehnološkog procesa prerade permeata, kao sporednog proizvoda dobijenog nakon ultrafiltracije mleka tokom proizvodnje feta sira i svežeg („mladog“) sira. Ispitana je mogućnost enzimske hidrolize laktoze u permeatu korišćenjem enzima β-galaktozidaze izolovanog iz Kluyveromyces lactis u koncentraciji 0,1, 0,3 i 0,5 g/100g na temperaturama 20º, 30º i 40 ºC. Praćene su promene sadržaja laktoze, D–galaktoze i D–glukoze u vremenskim intervalima tokom 60 minuta. Posebna faza istraživanja obuhvatila je matematičko modelovanje i kinetiku procesa hidrolize laktoze u permeatu pod dejstvom β –galaktozidaze i primenu hidrolizovanog permeata u proizvodnji mlečnih napitaka po odabranoj formulaciji. Predložen je tehnološki proces proizvodnje napitka na bazi hidrolizovanog permeata sa dodatkom voćnih baza. Utvrđeni su parametri kvaliteta i trajnosti napitaka tokom 60 dana skladištenja.Na temperaturi 40°C dodatkom enzima β -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.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°C) do preko 0,990 (na temperaturama 30°C i 40°C) pri koncentraciji enzima 0,1g/100g.Proizvedeni napici od hidrolizovanog permeata su delaktozirani i ne sadrže mlečnu mast. Od ukupnih šećera u svim napicima više od 50% čini glukoza: 50,16% - napitak šumsko voće, 50,42% - napitak pomorandža/šargarepa, 54,65% - napitak multivitamin, odnosno 55,13% - napitak crveno voće.Najveći sadržaj vitamina C nakon proizvodnje imao je napitak sa dodatkom voćne baze multivitamin 0,3972 mg/100g, zatim šumsko voće 0,2887 mg/100g i pomo-randža/šargarepa 0,1999 mg/100g.Najveću vrednost antioksidativne aktivnosti nakon proizvodnje pokazali su uzorci napitka sa multivitaminom i šumskim voćem. Tokom perioda skladištenja dolazi do smanjenja DPPH vrednosti. Najmanji pad je u napitku sa pomorandžom / šargarepom (smanjenje za 17%), a najveći u napitku sa šumskim 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.Generalno može se zaključiti da se prime-njenim tehnološkim procesom dobijaju napici stabilnog fizičko-hemijskog sastava tokom 60 dana skladištenja, visoke nutritivne i niske energetske vrednosti.
Valuation of the permeate as a by-product of the dairy industry is of great ecological, economic and technological importance.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 β-galactosidase isolated from Kluyveromyces lactis in a concentration of 0.1, 0.3 and 0.5 g / 100 g at a temperature of 20°, 30° and 40° 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 β-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.Addition of the enzyme β-galactosidase at a concentration of 0.1 g / 100 g for 60 minutes at a temperature of 40 ° 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.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° C) up to over 0.990 (at temperatures 30° C and 40° C) at a an enzyme concentration of 0.1g / 100g.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.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).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.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.

Nous avons étudié le mécanisme de sécrétion de Myxococcus xanthus, bactérie à gram-négatif, en utilisant comme sonde une protéine étrangère, la phosphatase hyperacide de Escherichia coli, qui est partiellement sécrétée chez M. Xanthus. Nous devions tout d'abord cloner son gène, appA, sous contrôle d'un promoteur inductible. En utilisant le gène lact comme gène rapporteur, nous avons comparé l'efficacité d'induction de deux promoteurs chez M. Xanthus, l'un endogène, le promoteur carQRs, inductible par la lumière, et l'autre exogène, le promoteur hybride tac, inductible par l'IPTG chez E. Coli en présence du gène répresseur lacIq: le système lacIq ptac est fonctionnel et inductible par l'IPTG chez M. Xanthus, mais le promoteur carQRs est plus fort chez M. Xanthus. Malgré de nombreux essais, nous ne sommes cependant pas parvenu à obtenir une souche de M. Xanthus chez laquelle la production de phosphatase hyperacide était inductible par la lumière. Nous avons alors clone le gène appA en aval du promoteur tac, puis obtenu une souche de M. Xanthus chez laquelle la production de phosphatase était inductible par l'IPTG. Par dosage, après induction, de l'activité appA dans les cellules et dans le surnageant, au cours du temps, et à trois niveaux d'induction différents, nous avons obtenu des cinétiques de sécrétion de la phosphatase hyperacide chez M. Xanthus dont l'analyse nous a permis de poser les bases d'un modèle de sécrétion chez M. Xanthus

Insulin regulated aminopeptidase (IRAP) was first identified in fat and muscle cells where it is believed to regulate GLUT4 translocation. It has since been found to be behind a variety of functions, many not yet fully understood. Preliminary research from Monash University suggested that IRAP may play a role in cellular senescence. Senescence is a term that describes arrested cell division and is a tumor repressive mechanism. Senescent cells have been shown to secrete, among other things, the growth hormone TGFβ1, which in turn plays an important role in the cell differentiation of fibroblasts to myofibroblasts. The potential link between IRAP and senescence was the basis of this work. Senescent fibroblasts from three different passages (n=3) in the BJ3 cell-line were cultured and treated with different IRAP inhibitors; ANG-4, AL06 and HFI-419 which were all compared to an untreated control group. They were marked with a β-galactosidase stain, a senescent cellmarker, and imaged. The study demonstrated that the IRAP inhibitors led to a certain decrease in % of senescent cells compared to the control groups. However, this reduction was not considered statistically significant. Similarly, inhibition of the enzyme did not indicate any influence over the differentiation of the cells. The lack of effect could be due to chance based on the low number of sample size, or the condition of the cells used in the trial as they were partially immortalized BJ fibroblasts well beyond the passage of their intended use. In order to further demonstrate an association between IRAP and senescence, further trials are required.
Insulin reglerad aminopeptidas (IRAP) introducerades till en början som ett markörprotein. Man har sedan dess funnit att den står bakom en rad olika funktioner, många ännu inte  fullt klarlagda. Preliminär forskning från laboratoriet i Monash University tydde på att IRAP kan ha en koppling till senescerande fibroblaster. Senescence är en term som beskriver upphörd celldelning och är en tumörrepressiv mekanism. Senescerande celler har påvisats utsekrera bland annat tillväxthormonet TGFβ1, som i sin tur spelar en viktig roll i celldifferentieringenav fibroblaster till myofibroblaster. Den potentiella kopplingen mellan IRAP och senescence låg som grund till detta arbete. Senescerande fibroblaster från tre olika kulturer (n=3) i BJ3-cellinjen odlades och behandlades med olika IRAP-inhibitorer; ANG-4, AL06 och HFI-419 som alla jämfördes med en kontrollgrupp. Därefter markerades de med en β-galaktosidas-markör, en markör för senescerande celler, och mikroskoperades. Studien påvisade att IRAP-inhibitorerna ledde till en viss procentuell minskning av senescerande celler jämfört med kontrollgrupperna. Dock bedömdes inte denna minskning som statistiskt signifikant i studien. Likväl fann man ingen procentuell minskning av differentierade fibroblaster. Hypotetiskt sett skulle man vilja se att reduktionen av senescerande celler motsvarade en nedreglering av TGFβ1-proteiner. Eftersom närvaron av TGFβ1 tros spela en ledande roll i celldifferentiering till myofibroblastfenotypen, bör den procentuella mängden differentierade cellerna minska med inhibitorbehandlingarna. Den bristande påverkan av enzyminhibitionen kan bero på en rad olika faktorer. Cellerna som användes under försökets gång var väl bortom deras brukliga användningscykel. För att vidare påvisa ett potentiellt samband mellan IRAP och senescence behöver vidare försök utföras.

Le nouveau plasmide circulaire, pKD1, isolé chez Kluvveromvces drosophilarum présente une organisation de son génome similaire à celle du plasmide 2 micron chez Saccharomyces cerevisiae, cependant ils ont très peu d'homologie au niveau de la séquence nucléotidique. L'analyse de la séquence nucléotidique de pKD1 montre qu'il a une taille de 4757 paires de bases, qu'il possède trois phases de lecture ouvertes (A, B et C) et une paire de répétitions inversées de 346 paires de bases. La molécule de pKD1 existe sous deux formes isomériques, ceci étant dû à une recombinaison intramoléculaire au niveau des répétitions inversées. La séquence protéique de la phase de lecture ouverte A a une homologie significative avec la protéine recombinase FLP du plasmide 2 micron. Le plasmide pKD1 a été transféré chez Kluyveromyces lactis où il peut être maintenu stablement sans pression de sélection. Nous avons développé un système de transformation efficace chez K. Lactis en utilisant des plasmides dérivés de pKD1. Cela nous a permis aussi de localiser les fonctions de pKD1 (origine de réplication, FLP, REP). De nombreux vecteurs (vecteurs de clonage, vecteurs d'expression, vecteurs pour la détection de promoteur et vecteurs de sécrétion) ont été construits en utilisant des séquences de pKD1 et différents marqueurs génétiques: le gène URA3 de S. Cerevisiae, le gène de résistance à la kanamycine de Tn903 et le gène lacZ d'E. Coli). Nous avons également développé un nouveau système de fusion de gènes à partir du gène de résistance à la kanamycine de Tn903, ce qui nous a permis d'analyser plusieurs promoteurs de S. Cerevisiae et K. Lactis
The new circular plasmid, pKD1 (or 1. 6µ), isolated from Kluyveromyces drosophlilarum, has a genome organization analogous to that of the 2 micron plasmid from Saccharomyces cerevisiae, although these plasmids share little sequence homology. Nucleotide sequence analysis of pKD1 revealed that this 4757 base-pair long plasmid contained three major open reading frames, A, B, and C, and a pair of inverted repeats of 346 base-pairs. The molecule exists in two isomeric forms generated by internal recombination at these repeats. The amino acids sequence encoded by open reading frame A has significant homology with the FLP recombinase of the 2 micron plasmid. The plasmid pKD1 can be transferred to Kluyveromyces lactis where it can be stably maintained. Using pKD1-derived recombinant plasmids, we developed an efficient transformation system for K. Lactis. This also allowed us to map the functions of pKD1 (replication origin, REP, FLP). A series of useful cloning vectors, expression vectors, promoter-probe vectors and secretion vectors were constructed using the pKD1 sequence and three different genetic markers: URA3 gene of S. Cerevisiae, kanamycin resistance gene of Tn903, and LACZ gene of E. Coli. A new gene fusion system was developed using the kanamycin resistance determinant of Tn903 as the indicator gene; in this way several promoters from S. Cerevisiae and K. Lactis were analysed

Die Entwicklung neuer humanmilchähnlicher, funktioneller und bioaktiver Lebensmittel (z. B. Trinkmilch oder Joghurt), die Oligosaccharide mit einer möglichen Gesundheitswirkung enthalten („Health Claim”), sind für die menschliche Ernährung von besonderem Interesse. Ziel dieser Arbeit war es daher, geeignete Verfahren zu entwickeln, um - unter Ausnutzung der Transferaseaktivität von β-Galactosidase - komplexe Galacto-Oligosaccharide aus Lactose zu synthetisieren und nicht-proteingebundene, komplexe milcheigene Oligosaccharide aus Kuhmilch aufzureinigen und zu charakterisieren. Zur Identifizierung und Quantifizierung der Oligosaccharide wurden zunächst hochempfindliche Analysenmethoden etabliert (siehe Kapitel 4.1). Da Oligosaccharide Minorkomponenten in der Milch sind, mussten diese von Fetten, Proteinen und Lactose abgetrennt werden. Die Entfettung erfolgte durch Zentrifugation in der Kälte. Die Proteinabtrennung war unter Verwendung einer 10kDa-Ultrafiltrations-Membran optimal (siehe Kapitel 4.1.1.1). Die Abtrennung der Lactose von den Oligosacchariden stellte die größte Herausforderung dar, da beide Stoffklassen zu den Kohlenhydraten gehören und sich nur geringfügig in ihren Molmassen unterscheiden. Des Weiteren liegt Lactose in Kuhmilch im ca. 1000-fachen Überschuss im Vergleich zu den Oligosacchariden vor. Beim Vergleich verschiedener Aufarbeitungsmethoden zur Lactoseabtrennung stellte sich die Festphasenextraktion an Aktivkohle (GCC-SPE) als am besten geeignet heraus. Mit dieser Methode wurde - im Gegensatz zur ebenfalls untersuchten Größenausschlusschromatographie - eine hohe Reproduzierbarkeit der Analyten erreicht. Während bei der Größenausschlusschromatographie das Kuhmilch-Oligosaccharid GalNAc-α-(1→3)-Gal-β-(1→4)-Glc nahezu vollständigen verloren ging, konnten nach GCC-SPEAufarbeitung fast 100 % des Analyten wiedergefunden werden (siehe Kapitel 4.1.1.2). Zur Charakterisierung der Oligosaccharide wurde eine hochauflösende Anionenaustauscherchromatographie mit pulsierender amperometrischen Detektion (HPAEC-PAD) etabliert. Parallel zur hochempfindlichen Detektion mittels PAD wurde zur direkten Strukturaufklärung von underivatisierten Zuckern eine Online-Kopplung an einen Massendetektor (IT-MS) aufgebaut. Einzelne Analyten konnten mit Hilfe von kommerziell erhältlichen Oligosaccharidstandards identifiziert und quantifiziert werden (siehe Kapitel 4.1.1.3). Als weitere Möglichkeit zur Quantifizierung der Oligosaccharide wurden photometrische Schnelltests entwickelt (siehe Kapitel 4.1.2). Zur Absicherung der HPAEC-PAD-Analysendaten sollten zukünftig weitere Analysenmethoden etabliert werden (z. B. enzymatischer Verdau der Oligosaccharide, Analyse der Monosaccharide nach Hydrolyse der OS, HILIC-HPLC). Die entwickelte HPAEC-PAD-Methode wurde zur Identifizierung und Quantifizierung von Oligosacchariden in Milchproben verschiedener Nutztierarten (siehe Kapitel 4.2.1) und in Produktströmen der milch- und molkeverarbeitenden Industrie (siehe Kapitel 4.2.2) verwendet. Die Untersuchungen von Milchproben unterschiedlicher Nutztierarten zeigten, dass jede Milch hinsichtlich der MOS einzigartig ist. Dies konnte anhand der verschiedenen, identifizierten MOS mit unterschiedlichen Konzentrationen belegt werden. Der höchste MOS-Gehalt konnte in Humanmilch detektiert werden, gefolgt von Kamelmilch, Schafsmilch, Ziegenmilch, Kuhmilch und Stutenmilch (siehe Kapitel 4.2.1). Die Untersuchungen der Produktströme der milch- und molkeverarbeitenden Industrie zeigten, dass es während der Lactosekristallisation und der Aktivkohlebehandlung zu Verlusten an MOS kommt. Bei anderen Prozessschritten, wie Entfettung, Proteinabtrennung, Aufkonzentrierung oder Käseherstellung, war kein Einfluss auf die MOS-Konzentrationen ersichtlich (siehe Kapitel 4.2.2). Die entwickelte Methode zur OS-Bestimmung mittels HPAEC-PAD könnte zukünftig auf die Analysen proteingebundener Oligosaccharide ausgedehnt werden [[Grey, 2009] und [Weiß, 2001]]. Zur Herstellung eines Oligosaccharid-angereicherten Lebensmittels wurden Galacto-Oligosaccharide enzymatisch - unter Ausnutzung der Transferaseaktivität von β-Galactosidase - aus Lactose synthetisiert (siehe Kapitel 4.3). Für die Entwicklung des Verfahrens wurden verschiedene β-Galactosidasen (Enzyme aus Kluyveromyces lactis, Aspergillus oryzae und Bacillus circulans) bei unterschiedlichen Reaktionsbedingungen getestet. Die höchste GOS-Ausbeute (19,8 Flächenprozent der HPAEC an Haupt-Trisaccharid, 40,5 % GOS) konnte mit Hilfe der β-Galactosidase aus B. circulans bei pH 4, 40 °C und 12 U/g erreicht werden. Dabei wurden v. a. Tri- bis Pentasaccharide gebildet. Das Hauptreaktionsprodukt war das Trisaccharid 4'-Galactosyllactose (4'-GL). Mit Hilfe der β-Galactosidase aus A. oryzae konnte bei pH 4,5, 40 °C und 12 U/g die zweitgrößte GOS-Ausbeute (14,2 % Haupt-Trisaccharid, 19,7 % GOS) synthetisiert werden. Dabei wurden v. a. Tri- und Tetrasaccharide mit dem Hauptreaktionsprodukt 6'-Galactosyllactose (6'-GL) gebildet. Die geringste GOS-Ausbeute (10,5 % Haupt-Trisaccharid, 3,0 % GOS) wurde mit der β-Galactosidase aus K. lactis bei pH 7, 40 °C und 12 U/g erreicht. Di- und Trisaccharide sowie ein geringer Anteil an Tetrasacchariden konnten dabei synthetisiert werden. Das Hauptreaktionsprodukt war hierbei das Trisaccharid 6'-GL. Außerdem konnte gezeigt werden, dass die milcheigenen Oligosaccharide während der Lactosehydrolyse mit den drei getesteten β-Galactosidasen weitgehend erhalten bleiben (siehe Kapitel 4.3.4). Neben den Galacto-Oligosacchariden, die nach enzymatischer Synthese direkt im Lebensmittel eingesetzt werden können, wurden milcheigene Oligosaccharide aus Kuhmilch aufgereinigt (siehe Kapitel 4.4.1). Zur Aufreinigung der MOS wurden Nanofiltrationsversuche in verschiedenen Maßstäben, mit unterschiedlichen Prozessparametern und diversen Membranen durchgeführt. Die im hohen Überschuss vorhandene Lactose wurde vor der Nanofiltration durch enzymatische Hydrolyse in ihre Monosaccharide gespalten, wodurch die Trennung von Monosacchariden und MOS verbessert wurde. Die Membran SR50/SR2 stellte sich für die MOS-Aufreinigung als am besten geeignet heraus. Mono- und Disaccharide konnten mit dieser Membran nahezu vollständig abgetrennt und MOS zu 42,1 % bis 52,4 % wiedergefunden werden. Das Verhältnis der Mono- und Disaccharide zu MOS konnte von ca. 1000:1 auf 18,5:1 zu Gunsten der MOS verändert werden. Der Anteil der Oligosaccharide am Gesamtzuckergehalt wurde von 0,1 % auf 5,1 % erhöht. Aus 40 kg hydrolysiertem Ultrafiltrations-Magermilchpermeat konnten 332,5 mg GalNAc-α-(1→3)-Gal-β-(1→4)-Glc, 414,1 mg 3'-SL und 91,5 mg 6'-SL gewonnen werden (Kapitel 4.4.1.4). Die durch Nanofiltration aufgereinigten, MOS-haltigen Proben sind für den Einsatz in einem potentiell funktionellen Lebensmittel geeignet. Da der Restlactosegehalt der synthetisierten, GOS-haltigen Proben und der aufgereinigten, MOS-haltigen Proben für weitere Analysen - wie orientierende Studien zur potentiell bifidogenen Wirkung - zu hoch war, erfolgte eine zweite Aufreinigung mittels präparativer Chromatographie an Aktivkohle (siehe Kapitel 4.4.2). Dadurch konnten bei den synthetisierten, GOS-haltigen Proben die Monosaccharide vollständig entfernt, der Restlactosegehalt auf unter 1 % am Gesamtzuckergehalt gesenkt und GOS aufgereinigt werden. Durch die Aktivkohle-Aufreinigung der durch Nanofiltration aufgereinigten, MOS-haltigen Proben konnten Mono- und Disaccharide von dem milcheigenen Oligosaccharid GalNAc-α-(1→3)-Gal-β-(1→4)-Glc sowie von den GOS - entstanden durch die vorherige Behandlung mit β-Galactosidase - abgetrennt werden. Die Sialyllactosen gingen dabei nahezu vollständig verloren. Auf Grund der vermuteten gesundheitsfördernden Wirkung der Sialyllactosen bedarf es weiterer Forschungsaktivitäten. Insbesondere ist eine Optimierung des Aufgabevolumens, der Konditionierung und der Wahl der stationären Phase wünschenswert. Mit den durch Aktivkohle aufgereinigten GOS-Lösungen, die weniger als 1 % Mono- und Disaccharide am Gesamtzuckergehalt enthielten, wurden orientierende Studien zur potentiell bifidogenen Wirkung durchgeführt (siehe Kapitel 4.5). Die orientierenden Studien mit Bifidobacterium longum ließen eine potentiell bifidogene Wirkung der untersuchten GOS-Lösungen erkennen. Diese GOS-Proben zeigten dabei eine stärkere bifidogene Wirkung als die bifidogene Referenzsubstanz Lactulose und der Vivinal®GOS-Sirup. Zukünftig sollten die Proben, die nach Beendigung der bakteriellen Reaktion gewonnen wurden, mittels HPAEC-PAD analysiert werden. Dadurch könnte der Kohlenhydratabbau bzw. die Bildung organischer Säuren untersucht werden. Der zeitliche Verlauf sowie der Einsatz anderer Mikroorganismen - wie Lactobazillen und Clostridien - könnten ebenfalls untersucht werden. Andere orientierende Studien, wie antiinflammatorische Tests, wären bei der weiteren Charakterisierung der Oligosaccharide hilfreich. Die Herstellung eines Oligosaccharid-angereicherten Lebensmittels erfolgte im Labormaßstab unter Zusatz der potentiell bifidogenen GOS-Lösung, die mit Hilfe von β-Galactosidase aus K. lactis synthetisiert wurde. Die Art der Erhitzung des Lebensmittels - Pasteurisierung vs. Hocherhitzung - hatte keinen Einfluss auf die Zuckerzusammensetzung und die Zuckergehalte. Während einer anschließenden sechswöchigen Lagerung in der Kälte konnte kein Abbau der Kohlenhydrate detektiert werden. Als Ausblick für weitere Forschungen ist die Bestätigung der potentiell bifidogenen Wirkung der MOS-haltigen Proben von primärer Bedeutung. Anschließend könnten Studien mit funktionellen, MOS-haltigen Lebensmitteln durchgeführt werden. Dabei sollte versucht werden, ein humanmilchähnliches Lebensmittel mit ca. 20 % sauren und ca. 80 % neutralen Oligosacchariden herzustellen. Die mittels Nanofiltration aufgereinigten, MOS-haltigen Proben könnten als saurer Zusatz und die enzymatisch synthetisierten, GOS-haltigen Proben als neutraler Zusatz verwendet werden. Des Weiteren sollte eine Überprüfung der potentiell bifidogenen Wirkung sowie eine sensorische Prüfung des hergestellten Lebensmittels durchgeführt werden. Sobald MOS durch Nanofiltration bzw. GOS mittels enzymatischer Synthese im großtechnischen Maßstab aufgereinigt bzw. hergestellt werden können, sind Humanstudien zur Bestätigung der Wirksamkeit von milcheigenen Oligosaccharide bzw. der Galacto-Oligosaccharide möglich. Im Rahmen der vorliegenden Arbeit konnten potentiell funktionelle Lebensmittelinhaltsstoffe (enzymatisch synthetisierte, GOS-haltige Lösungen) und Lebensmittelinhaltsstoffe, deren vermutete Funktionalität noch nicht bestätigt wurde (durch Nanofiltration aufgereinigte, MOS-haltige Lösungen), hergestellt werden. Unter Verwendung der in dieser Arbeit gewonnenen Erkenntnisse und der Erfüllung der im Ausblick geschilderten Bedingungen, ist eine großtechnische Produktion eines funktionellen, Oligosaccharid-angereicherten Lebensmittels möglich.

Tese de mestrado, Controlo de Qualidade e Toxicologia dos Alimentos, Universidade de Lisboa, Faculdade de Farmácia, 2012
A intolerância à lactose é a mais comum a um hidrato de carbono e pode afectar pessoas de todos os grupos etários, apesar de ocorrer com mais frequência em idades avançadas. A intolerância à lactose é causada pela deficiência em lactase (β-galactosidase), a enzima que digere o açúcar do leite, a lactose. Neste trabalho pretendeu-se estudar a enzima β-galactosidase, proveniente de Kluyveromyces lactis, sob diferentes formas e em diferentes condições experimentais, através da variação da concentração de enzima, concentração de substrato padrão, o-nitrofenol-β-ᴅ-galactoside, e da variação temperatura. Foram depois realizados ensaios na matriz alimentar, o leite, uma vez que o pH óptimo desta enzima, 6,8, é semelhante ao pH do leite. Para tal, foi utilizada a enzima Lactozym 2600L, disponibilizada comercialmente pela Sigma-Aldrich® sob a forma de suspensão líquida e a enzima imibond galactosidase SPRIN®, disponibilizada comercialmente de forma imobilizada numa resina amino-acrílica pela SPRIN Tecnhologies®. Foi também realizada a imobilização da enzima Lactozym 2600L em suporte de alginato de cálcio. De todas as formas enzimáticas testadas, a imibond galactosidase SPRIN® foi a que apresentou melhores resultados, nomeadamente a nível da estabilidade operacional nos ensaios de reutilização. No final foram utilizados os valores optimizados na matriz alimentar, o leite, de modo a testar a actividade hidrolítica da enzima sobre a lactose, não se conseguindo obter um grande rendimento de conversão nesta reacção, sugerindo a necessidade da realização de mais estudos e ensaios nesta promissora área da biotecnologia.
Lactose intolerance is the most common one carbohydrate and can affect people of all age groups, although occur more frequently in elderly persons. Lactose intolerance is caused by a deficiency of lactase (β-galactosidase), the enzyme that digests the milk sugar lactose. This work aimed the study of the enzyme β-galactosidase, obtained from Kluyveromyces lactis in different forms and in different experimental conditions, by varying the enzyme concentration, standard substrate concentration, o-nitrophenol-β-ᴅ-galactoside, and temperature variation. Assays were then performed in the food matrix, milk, since the pH optimum of this enzyme, 6.8, is similar to pH of milk. To do this, the enzyme used was Lactozym 2600L, commercially available from Sigma-Aldrich ® in the form of liquid suspension and enzyme imibond galactosidase SPRIN®, available commercially immobilized on an amino-acrylic resin by SPRIN® Tecnhologies. It was also performed immobilization of de the enzyme Lactozym 2600L in a calcium alginate support. Of all the enzyme forms tested, the imibond galactosidase SPRIN® showed the best results, particularly in terms of operational stability in the reuse assays. At the end, values optimized were used in the food matrix, milk, in order to test the hydrolytic activity of the enzyme on the lactose, but was not possible to obtain a high conversion yield, suggesting a need for further studies and tests on this promising area of biotechnology.

碩士
國立臺灣大學
生化科學研究所
93
Deinococccus sp. NTU-1079 is isolated from Zhi-Sung hot spring in Taitung and found thermophilic as well as resistant to ionizing radiation. The gene of β-galactosidase from Deinococccus sp. NTU-1079 is cloned and the recombinant β-galactosidase is overexpressed in Escherichia coli, then simply purified by Ni-NTA column. The extracellular α-galactosidase is purified sequentially by DEAE sepharose, Superose 6 HR and HiTrap Q chromatography to homogeneity Characterization experiments show that the two enzymes are stable under 50℃ for 3 h, and have the highest activities at neutral pH around 60 ~ 65℃. They are highly resistant to X-ray, under exposure to 2.58 KGy for 8 h, showing that the α-galactosidase and the β-galactosidase have remaining activity about 25% and 90%, respectively. Their Km and Vmax values for different substrates are also obtained. Furthermore, we find that the β-galactosidase belongs to glycoside hydrolase family 42 based on primary sequence alignment, and possesses similar structure to 4/7 superfamily enzymes according to the secondary and tertiary structure predictions. The tertiary structure of the β-galactosidase is built by computer modeling based on the known structure of β-galactosidase from Thermus sp. A4 as a template and the thermostability of the β-galactosidase may come from the formation of hydrogen bonds between subunits. The quaternary structure of the β-galactosidase is examined by gel filtration and sedimentation equilibrium and shows as dimer and trimer, respectively. The transgalactosyl properties of these enzymes are applied to synthesize oligosaccharide derivatives as lectin-ligand probes. Using characteristics of thermostability, organic solvent-resistance, radiation resistance and specific catalytic mechanism of the enzymes, lectin-ligand probes can be established rapidly by the chemoenzymatic method.

碩士
國立雲林科技大學
化學工程與材料工程研究所
95
Galacto-oligosaccharides (GOS) find particular use in promoting the growth of bifidobacteria in the lower part of the human intestine and have become a popular food additive for health purpose. GOS can be produced from lactose by enzymatic hydrolysis and transgalactosylation reaction using β-galactosidases (EC 3.2.1.23). Lactase-N10 from Bacillus circuians was separated and purified into three different enzyme groups. Each group of purified β-galactosidases was used to produce GOS in the batch reactor and the purities of GOS from the three enzyme groups were compared. The results show that, in the first step, β-galactosidases was concentrated from the crude enzyme (specific activity was 274.6 U/mg) by ammonium sulfate precipitation of 10 ~ 50 % (w/v), the specific activity of the concentrated product was 480.1 U/mg and the recovery of the enzyme activity was 95.4 %. In the second step, β-galactosidases was further concentrate by QAE Sephadex A-50 ion-exchange in the batch mode, the specific activity of the further concentrated product was 502.9 U/mg and the recovery of the enzyme activity was 65.0 %. In the third step, β-galactosidases was purified by Sephacryl S-200HR gel filtration chromatography. Two fractions were collected to get the two groups of purifiedβ-galactosidases (β-gal Ι and β-gal ΙI). The specific activities were 2317.4 U/mg and 3360.1 U/mg, respectively, and the recoveries of the enzyme activity were 54.3 % and 31.3 %, respectively. Finally, the two groups of purifiedβ-galactosidases and the crude enzyme were used to produce GOS in the batch reactor, the operating conditions were concentration of 10 %(w/v) for both the lactose solution and enzyme solution, volume of 10 ml for the reaction solution, enzyme/substrate ratio of 1:1000 (v/v), reaction temperature of 45 ℃, and pH 6. The results show that, the crude enzyme reached the highest GOS purity of 16.89 % (w/w) at 120 min reaction time, β-gal Ι enzyme reached the highest GOS purity of 16.32 %(w/w) at 20 min reaction time, and β-gal ΙI enzyme reached the highest GOS purity of 32.10 %(w/w) at 60 min reaction time. The purified enzyme β-gal ΙI, due to its relatively strong function of transgalactosylation and the relatively weak function of hydrolysis, can produce the highest purity of GOS.

碩士
國立雲林科技大學
工業化學與災害防治研究所
93
Galacto-oligosaccharides (GOS), due to their particular use in promoting the growth of bifidobacteria in the lower part of the human intestine, inhibiting microbe in intestine, stimulating the peristalsis of the intestine and promoting to empty the bowels, have become a popular food additive for health purpose. GOS can be produced from lactose by enzymatic hydrolysis and transgalactosylation reaction using β-galactosidase. Enzyme immobilization provides many important advantages over use of enzyme in soluble form, namely, higher thermal stability, enzyme reusability, and continuous operation. The objective of this study is to develop an efficient and easy-to-scale-up technique for the immobilized enzyme reaction, by which the GOS can be continuously produced with high purity and high productivity. The results show that, when using chitosan as the support, the optimal concentrations used in the immobilization process were 0.1%(w/v) polyethylene glycol solution, 10%(v/v) glutaraldehyde solution, and 8 mg/ml enzyme solution. The immobilization amount was above 5 mg protein/g support and the activity was above 160 Units/g support or 78 Units/g support when taking lactose or glucose plus galactose as a basis. In addition, when using Amberlite IRC-50 as the support, the optimal concentrations used in the immobilization process were 100 mg/ml polyethylenimine solution, 10%(v/v) glutaraldehyde soluion, and 8 mg/ml enzyme solution. The immobilization amount was above 6 mg protein/g support and the activity was above 131 Units/g support or 89 Units/g support when taking lactose or glucose plus galactose as a basis. For producing GOS using free enzyme in the batch reactor, the optimal conditions were found to be 30%(w/v) lactose solution, 0.75% enzyme/substrate ratio , 35℃ reaction temperature, pH 4, and 40 min reaction time, from which the GOS concentration was about 24%(w/w). In addition, the thermal stabilities of the free enzyme and the two immobilized enzymes were compared based on the first-order deactivation rate measurement in the batch reactor. It was found that the immobilized enzymes had better thermal stability than that of the free enzyme and the Amberlite IRC-50 was chosen to be used in the continuous production of GOS in the packed bed reactor due to its best thermal stability and mechanical strength. For continuously producing GOS using immobilized enzyme in the packed bed reactor, the optimal conditions were found to be 30%(w/v) lactose solution in pure water, 35℃ reaction temperature, and 3.33 hr-1 space velocity, from which the GOS concentration was about 19%(w/w) and the productivity was about 140 g oligosaccharides/litre-hr. Also, after 16 days of long-term operation, the purity and the productivity were almost retained. Thus, after properly scaling-up the process, it can be used economically and efficiently for continuous production of GOS.

Tese de Doutoramento em Engenharia Química e Biológica - Área de Conhecimento Engenharia Enzimática e das Fermentações
Esta tese de Doutoramento é o resultado de um projecto de colaboração entre a Universidade Federal de Pernambuco, Brasil e a Universidade do Minho, Portugal, financiado pelo Programa Alßan de bolsas de estudo de alto nível destinado à América Latina. Lactases ou β-galactosidases (E.C. 3.2.1.23) são enzimas com um elevado interesse industrial quer na conversão da lactose em galactose e glicose quer na síntese de oligossacarídeos. O seu uso tem sido recomendado para hidrolisar a lactose no leite consumido por aqueles indivíduos com intolerância à lactose, na manufactura de sorvete e na produção de galactooliossacarídeos (GOS), compostos que se inserem nos denominados alimentos funcionais (pré-bióticos). A imobilização desta biomolécula em suportes de baixo custo, mediante procedimentos de fácil execução, pode potenciar a eficiência catalítica do derivado enzimático por causa da melhor estabilidade ao pH e temperatura, proporcionando deste modo, menores custos operacionais e aumentando suas aplicações biotecnológicas em indústrias de alimentos. O principal objectivo do trabalho desenvolvido foi avaliar a utilização de β-galactosidase imobilizada em diferentes matrizes insolúveis em água tanto na hidrólise da lactose como na síntese de GOS. No decorrer do trabalho, foram usados quatro suportes magnéticos: (1) polisiloxano-Álcool Polivinílico magnético – mPOS-PVA; (2) magnetita revestida com polianilina – MAG-PANI; (3) polisiloxano revestido com polianilina – POS-PANI e (4) Dacron magnetizado. β-Galactosidase de duas origens diferentes (Kluyveromyces lactis e Aspergillus oryzae) foram usadas. Todas as matrizes investigadas foram adequadas para a imobilização de β-galactosidase e para a produção de GOS usando lactose como substrato. A capacidade de formação de GOS pela enzima não foi afectada pela imobilização nos diferentes suportes magnéticos, não se tendo observado diferenças na cinética reaccional de síntese entre a enzima livre e a enzima imobilizada nos diferentes suportes. A caracterização físico-quimica dos suportes contendo a enzima imobilizada permitiu confirmar a ausência de limitações difusionais à transferência de massa. Verificou-se também que a produção de GOS, a diferentes temperaturas 30 – 60ºC, foi praticamente inalterada, tanto para a enzima livre quanto para a imobilizada. Comparando a eficiência de imobilização dos diferentes suportes, verificou-se que a maior retenção de actividade enzimática de hidrólise e de síntese foi obtida com a enzima imobilizada em Dacron magnetizado. Foi também desenvolvido um modelo matemático que descreve adequadamente as reacções de hidrólise da lactose e síntese de GOS e efectuada a caracterização química, física e estrutural dos suportes MAG-PANI e POS-PANI.
This PhD thesis is the result of a collaboration project between the Universidade Federal de Pernambuco, Brazil, and the Universidade do Minho, Portugal, financed by the Programme Alßan, a high level scholarship programme specifically addressed to Latin America. Lactases or β-galactosidases (E.C. 3.2.1.23) are enzymes with an increasing industrial importance for lactose hydrolysis and for oligosaccharides synthesis. Its use has been recommended for the hydrolysis of lactose in milk consumed by lactose intolerant individuals, for ice-cream manufacture and in the production of galactooligosaccharides (GOS), compounds known as functional foods (prebiotics). The immobilization of β-galactosidase onto low-cost matrices by ease procedures can enhance the catalysis efficiency of the enzymatic derivatives because of the greater stability to pH and temperature, providing thus lower operational costs and increasing their applications in food industry biotechnology. The main objectives of the developed work were to evaluate the immobilization of β-galactosidase onto different water insoluble matrices both for lactose hydrolysis and GOS synthesis. Four magnetic matrices were used for enzyme immobilization: (1) polyvinyl alcohol polysiloxane-magnetic - mPOS-PVA; (2) magnetite coated with polyaniline - MAG-PANI; (3) polysiloxane coated with polyaniline - POS-PANI and (4) ferromagnetic Dacron. β-galactosidase from two different origins (Kluyveromyces lactis and Aspergillus oryzae) were used. All the investigated matrices were suitable for the β-galactosidase immobilization and the GOS production as well, using lactose as substrate. The ability of GOS formation by the enzyme was not affected by the immobilization on the different magnetic matrices and no differences on the kinetics of GOS synthesis were observed between immobilized and free enzymes. The physic-chemical characterization of the supports with the immobilized enzymes allowed the conclusion that diffusional mass transfer limitations were not a concern. It was observed that GOS production in the temperature range of 30 - 60 º C was unchanged for both free and for immobilized enzyme. In what concerns β-galactosidase immobilization efficiency of the different supports, it was conclude that magnetized Dacron allowed for the highest activity retention both for hydrolysis and synthesis. Furthermore, a mathematical model describing lactose hydrolysis and GOS synthesis was developed and a detailed chemical, physical and structural characterization of the MAG-PANI and POS-PANI supports was done.
Program Alban - E05D057787BR.

Dissertação de mestrado em Bioengenharia
β-galactosidases (EC 3.2.1.23), also known as lactases, are a family of enzymes that are able to catalyze two different reactions, namely the hydrolysis of lactose and transgalactosylation. The hydrolytic activity is commonly applied in the food industries for reducing the lactose content on dairy products, but it is also important to prevent lactose crystallization problems and to increase the sweetening capacity. Transgalactosylation reactions had been used to synthesize lactose derivatives, such as galactooligosaccharides (GOS), lactulose and lactosucrose. These compounds are classified as prebiotics, which are functional food ingredients, are not digested on the gastrointestinal (GI) tract and are able to stimulate the growth or activity of health-promoting bacteria. The sources of β-galactosidases are extensively distributed in nature, namely in microorganisms, plants and animal organs. Aspergillus lacticoffeatus was chosen for this work as a potential β-galactosidase producer based on preliminary chromogenic tests performed in agar plates. Herein, additional studies carried out under submerged fermentation conditions confirmed the presence of β-galactosidase in the fermentation broth, as well as in the cell extract obtained after cell disruption by ultrasounds. Therefore, this work represents the first time that A. lacticoffeatus is described as a β-galactosidase producer. The enzyme production was evaluated in different fermentation media: synthetic medium composed by lactose (20 g/L), yeast extract (4 g/L), peptone (4 g/L) and salts; and alternative fermentation media with some industrial by-products such as cheese whey and corn steep liquor (CSL). The highest values of extracellular enzymatic activity (444 U/L) were obtained using the synthetic medium. The extracellular enzyme presented a molecular weight between 70–150 kDa and optimal pH and temperature in the range 3.5–4.5 and 50–55 ºC, respectively. The effect of some metal ions (Na+, K+, Li+, Ba2+, Fe2+, Mg2+, Zn2+, Mn2+, Co2+ and Cu2+), detergents (Triton, SDS and Tween), additives (EDTA, PMSF and ascorbic acid) and sugars (glucose, fructose and galactose) on the enzymatic activity was also evaluated. Finally, the potential application of the enzyme for the synthesis of lactose-based prebiotics was studied and it was demonstrated that the β-galactosidase from A. lacticoffeatus is able to catalyze the transfer reactions involved in the formation of lactulose and GOS.
As β-galactosidases (EC 3.2.1.23), também conhecidas como lactases, são uma família de enzimas capazes de catalisar dois tipos diferentes de reações, nomeadamente a hidrólise da lactose e a transgalactosilação. A atividade hidrolítica é frequentemente aplicada na indústria alimentar na redução do conteúdo de lactose em produtos lácteos, mas é também importante na prevenção da cristalização da lactose e no aumento da capacidade adoçante. As reações de transgalactosilação têm sido usadas na síntese de derivados de lactose, tais como galacto-oligossacáridos (GOS), lactulose e lactosucrose. Estes compostos são classificados como prebióticos, ou seja, ingredientes de alimentos funcionais que não são digeridos no trato gastrointestinal e são capazes de estimular o crescimento ou atividade de bactérias benéficas para a saúde. As fontes de β-galactosidases estão amplamente distribuídas pela natureza, nomeadamente em microrganismos, plantas e órgãos animais. Aspergillus lacticoffeatus foi escolhido para este trabalho como potencial produtor de enzima tendo em conta os resultados preliminares de testes cromogénicos realizados em placas de agar. Neste trabalho, estudos adicionais conduzidos sob fermentações submersas confirmaram a presença de β-galactosidase no caldo da fermentação, assim como no extrato celular obtido após rutura celular por ultrassons. Desta forma, A. lacticoffeatus é aqui pela primeira vez descrito como um produtor de β-galactosidase. A produção da enzima foi avaliada em diferentes meios de fermentação: meio sintético composto por lactose (20 g/L), extrato de levedura (4 g/L), peptona (4 g/L) e sais; e meios alternativos constituídos por resíduos industriais como o soro de queijo e corn steep liquor (CSL). Contudo, os valores mais elevados de atividade enzimática extracelular (444 U/L) foram obtidos usando o meio sintético. A enzima produzida extracelularmente apresentou um peso molecular entre 70 e 150 kDa e o pH e temperaturas ótimos num intervalo entre 3.5–4.5 e 50–55 ºC, respetivamente. O efeito de iões metálicos (Na+, K+, Li+, Ba2+, Fe2+, Mg2+, Zn2+, Mn2+, Co2+ e Cu2+), detergentes (Triton, SDS e Tween), aditivos (EDTA, PMSF e ácido ascórbico) e açúcares (glucose, frutose e galactose) na atividade enzimática foi também avaliado neste trabalho. Finalmente, a potencial aplicação da enzima na produção de prebióticos derivados de lactose foi estudada, tendo-se demonstrado que esta β-galactosidase de A. lacticoffeatus é capaz de catalisar as reações de transferência envolvidas na formação de lactulose e GOS.

碩士
明志科技大學
生化工程研究所
96
In this study, the commercial STREAMLINE DEAE adsorbent was used to investigate the adsorption and desorption characteristics b-D-galactosidase (b-gal, EC 3.2.1.23) in the crude cell homogenate. In a series of batch and packed bed operations, the optimal adsorption and elution conditions for b-gal were obtained. These experiments included a series of operating parameters such as the choice of absorption pH, the effect of whole cells on the binding capacity, the experiments equibrium adsorption isotherm and uptake rate were also investigated. The effects of ionic strength, salt concentration, and linear velocity on the elution process were carried out in a small packed bed. The results were used to assess the feasibility of using stirred fluidized bed chromatography for direct recovery of b-gal from different operating conditions in highly crude cell homogenates. The recovery yield for b-gal was approximately 45.07% with a purification factor of 2.07 by using 0.5 M NaCl as an eluent at a linear velocity of 150 cm/h.

碩士
國立臺灣大學
食品科技研究所
86
Galactooligosaccharides (GalOS) can be produced from lactose by an enzymat ic transgalactosylation reaction.It has been shown that GalOS can induce the growth of bifidobacterium, a healthy microbe, in the intestine of humans. The m ain goal of this study was to evaluate the potential of manufacturing low-lact ose- and high-GalOS-milk (LLHGM) products using b-galactosidase. Two appro aches for manufacturing LLHGM products were considered in this study. Thefirs t mthod used b-galactosidase to transfer the lactose in milk to GalOS directl y. The second method applied ultrafiltration techniques to separate the lactos e in milk from other big moleculars, such as proteins, and then transferred th e lactose in the permeate to GalOS by b-galactosidase, and finally added the p ermeate back to the retentate. Our results indicated that the optimal cond itions for the direct enzymatic approach for producing LLHGM were found to be 16.7% lactose concentration, 5.1% enzyme/substrate ratio at 47oC for 1.4hr, wi th the maximum yield of 22.8% GalOS and the residue of 34.7% lactose. Neverth eless, the optimal conditions for the enzymatic reaction in the permeate was 2 5.3% lactose concentration, 6.7% enzyme/substrate ratio at 50oC for 3.5hr, wit h the maximum yield of 31.1% GalOS and the residue of 35.3% lactose. Alth ough the yield of GalSO under the enzymatic reaction in the permeate was signi ficantly higher than that under direct enzymatic reaction in milk, the yield o f GalSO calculated as a ratio to the total lactose in milk made no significant difference between the two cases. The results further indicated that when mi lk was processed by ultrafiltration, the yield of GalSO in terms of the total lactose depended upon how well the system can separate the lactose. In other words, the more lactose in the permeate, the higher the yield of GalSO. Howev er, the cost for the operation of ultrafiltration is expensive, particularly w hen high efficiency in separating the lactose is desired. Therefore, it requir es furtherinvestigation to determine whether manufacturing LLHGM products by a pplying ultrafiltration techniques is economically feasible.

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.

碩士
大同大學
生物工程學系(所)
93
Galactooligosaccharides (GOS) have been received increasing interest, because they can promote the proliferation of bifidobacteria and lactic acid bacteria in the human intestine and thus enhance the human health. GOS is the collective name of oligosaccharides constructed by 2-5 glycosyl moieties, mainly galactoside. Commercial GOS are produced from lactose by the transgalactosyl catalytic activity of β-galactosidase. Because of the enzymatic behavior, commercial GOS consisted of large amount of glucose and lactose. Such product not only was a low-content GOS, but also was not available for the lactose-intolerant people. The present study engaged in the enhancement in GOS yield by a mixed-enzyme system and the increase in GOS content by the yeast fermentation. Among the β-galactosidases from Kluyveromyces lactics, Aspergillus oryzae and Huang-Tai Company, the enzyme from Huang-Tai Company resulted in the highest concentration and yield of GOS. In the mixed-enzyme system, in which β-galactosidase and glucose oxidase were used, the glucose byproduct was converted into calcium gluconate precipitate and the GOS yield was increased from 25% to 28%. During the fermentation of low-content GOS by Kluyveromyces marxianus, the digestible sugars including glucose and lactose were depleted and the GOS content was increased from 25% to 96%, on a dry weight basis.

Single molecule studies of enzymes have revealed that nominally identical individual enzyme molecules are functionally heterogeneous. Different individual molecules exhibit different catalytic rates under identical conditions, and individual enzyme molecules show fluctuating rates over broad timescales. The structural basis and the biological sources for such heterogeneity remains poorly understood. Herein, studies are presented of the β-galactosidase from Escherichia coli, using capillary electrophoresis with laser-induced fluorescence (CE-LIF), to investigate the sources of catalytic heterogeneity at the single molecule level. Limited proteolysis as a possible source for single molecule heterogeneity, and for the changes in activity of a population of individual molecules over time, was investigated by inducing enzyme expression in two E.coli strains in the presence of a broad spectrum of protease inhibitors. The effect of protease inhibitors was found to be limited. β-Galactosidase was expressed from a lacZ linear template from two different E. coli strains using an in vitro protein expression system to determine if in vitro synthesized enzyme was identical to its in vivo counterpart. In vitro synthesized enzyme was found to be less active than in vivo sources. The differences were attributed to deficient N-terminal methionine removal and the higher rates of translation error associated with in vitro protein synthesis. Single molecule separations revealed that individual molecules of β-galactosidase were electrophoretically distinct, and that the electrophoretic heterogeneity was independent of source of enzyme, method of measurement, or of capillary coating. Electrophoretic modeling indicated that slight variation of hydrodynamic radius is the most likely source of electrophoretic mobility heterogeneity. The extent of single molecule catalytic variation was reduced in a mutant with a hyperaccurate translation phenotype implying that translation error is a source of the heterogeneity. Streptomycin-induced translation error reduced average activity, but did not lead to an increase in catalytic heterogeneity. No relationship between translation error and electrophoretic heterogeneity was observed. A novel CE-LIF assay was developed for the continuous monitoring of the catalytic activity and electrophoretic mobility of individual β-galactosidase molecules. Thermally-induced catalytic fluctuations were observed suggesting that individual enzyme molecules were capable of conformational fluctuations that supported different catalytic rates.

博士
國立臺灣大學
食品科技研究所
94
β-Galactosidase produced by some microorganisms, in addition to catalyze the hydrolysis of lactose, exhibits a transgalactosylation activity (TGase activity) which can catalyze the formation of galactooligosaccharides (GOS), a prebiotics, from lactose. In this study, experiments were conducted to investigate the production of β-galactosidase which processes transgalactosylation activity from Bifidobacterium. In addition, purification and characterization of β-galactosidase from B. longum BCRC 15708 as well as the synthesis of GOS with this enzyme were performed. β-Galactosidase production by Bifidobacterium strains, including B. longum BCRC 14634, 15708 as well as B6, B. breve BCRC 11846, B. bifidum BCRC 14615, B. adolescentis BCRC 14608, and B. infantis BCRC 14633, was first carried out in flask, with B. longum BCRC 15708 showing the highest production of β-galactosidase with the highest specific activity. Further study with B. longum BCRC 15708, it was first found that lactose and yeast extract, respectively, were the best carbon source and nitrogen source for β-galactosidase production. After 16 hrs cultivation, a maximum β-galactosidase activity of ca 18.6 U/ml was obtained in medium containing 4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO4•7H2O, 0.03% L-cysteine, and with the initial pH value and temperature controlled at 6.5 and 37℃, respectively. β-Galactosidase from B. longum BCRC 15708 was purified by procedures including Q Fast-Flow chromatography and Superose 6 HR gel chromatography. These steps resulted in a purification of 15.7-fold, a yield of 29.3% and a specific activity of 168.6 U mg-1 protein. The molecular weight was 357 kDa as determined from Native-PAGE. Using o-nitrophenyl-β-D-galactopyranoside (ONPG) as substrate, the pH and temperature optimum of the purified β-galactosidase were 7.0 and 50℃, respectively. The Km and Vmax were 0.85 mM and 70.67 U/mg, respectively. Monovalent cations (Na+ and K+) stimulated the enzyme up to 10-fold, whereas Fe3+ and bivalent cations (Fe2+, Co2+, Cu2+, Ca2+, Zn2+, Mn2+, and Mg2+) in the concentration of 1 mM inhibit β-galactosidase activity. Furthermore, galactose, lactose, and fructose would also inhibit the enzyme activity. B. longum BCRC 15708 grown in a jar fermenter showed that inoculum size, cultivation temperature, the pH of medium and agitation speed all would affect β-galactosidase production. A maximum β-galactosidase activity of 36.7 U/ml and a maximum transgalactosylation activity of 0.49 U/ml were achieved after 10 hr of fermentation with 20% inoculum of the test organism into a medium containing 4% lactose and 3.5% yeast extract, and with the pH of medium, cultivation temperature and agitation speed controlled at 6.5, 37℃ and 100 rpm, respectively. Two types of GOS, tri- and tetrasaccharides, were formed by transgalactosylation catalyzed by β-galactosidase from B. longum BCRC 15708, while trisaccharides were the major type of GOS formed. A maximum yield of 30.1% (w/w) GOS could be achieved from 40% lactose solution at 45℃ and pH 6.8 when the lactose conversion was 57.8%. The corresponding productivity of GOS was 13.9 g/l•h. Moreover, when more than 15% of galactose or glucose was added to reaction mixture containing 40% lactose, transgalactosylation reaction was markedly inhibited by 17.3-35.8%.