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.

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.

In this study, molecular cloning studies were performed on the &amp / #945 / -galactosidase genes of Aspergillus fumigatus IMI 385708. This organism is an opportunistic saprophytic fungus and a human pathogen, mainly affecting immunocompromised patients. A. fumigatus is a thermotolerant fungus and can efficiently produce thermostable &amp / #945 / -galactosidase. Two different cloning strategies were undertaken in this study. A. fumigatus cDNA library, prepared previously, was screened with three different probes. No net results were obtained from these screenings. However, the DNA probes used were shown to be homologous to the &amp / #945 / -galactosidase gene (agl1) of Trichoderma reesei. After the completion of the genome project of A. fumigatus, regions with homology to &amp / #945 / -galactosidase genes were searched on the genome of A. fumigatus. PCR-based cloning studies were performed by designing specific primers for these regions. Two &amp / #945 / -galactosidase genes, namely aglA and aglB were amplified with these specific primers, sequenced, and ligated to vector pUC19. The recombinant plasmid was then used to transform E. coli XL1 Blue MRF&rsquo / cells. aglB gene consists of an open reading frame of 1684 bp containing six introns. The gene encodes a protein of 447 amino acids with a signal sequence of 22 amino acids and four N-glycosylation sites. aglA gene has an open reading frame of 1599 bp without introns. The gene encodes a protein of 532 amino acids with a putative signal sequence of 21 amino acids and four putative N-glycosylation sites. Cloning of &amp / #945 / -galactosidase genes represents a first step towards the high level expression of these enzymes in a GRAS host.

The enzyme, &amp / #946 / -galactosidase (E.C.3.2.1.23) has been used for dairy industry for removing lactose from milk and milk by-products. In this study, three strains namely L. plantarum NCIMB 1193, L. plantarum DSM 20246 and L. plantarum E081 were used for &amp / #946 / -galactosidase release by sonication method. The peak of the total enzyme activity was found to be corresponding to late logarithmic or early stationary phase of all strains. As a disruption method sonication was used for the release of &amp / #946 / -galactosidase. Meanwhile, the sonication time was optimized for each strain. The peak of the enzyme activity was observed between 210 seconds and 270 seconds of sonication period. It was also found that sonication did not decrease the viability of L.plantarum NCIMB 1193 significantly. Liquid nitrogen cell disruption method was also used to compare the results with those obtained by sonication method. For characterization &amp / #946 / -galactosidase, cell-free crude extract of sonicated cell culture of L.plantanrum NCIMB 1193 was used. Optimum pH found as 7.2, and optimum temperature range was found between between 350 C to 400 C. Km and Vmax values were found as 3.47 mM and 1.721 (&amp / #956 / mol / min per mg protein) respectively from Lineweaver-Burk plot. Km and Vmax values were found as 4.064 mM and 1.863 (&amp / #956 / mol / min per mg cell-free crude extract) respectively from Eadie-Hofstee plot. The number of ligand binding sites (napp) on a molecule of &amp / #946 / -galactosidase was found as 1.03 which indicates that the number of ligand binding sites on the enzyme is one.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed June 23, 2008). Includes bibliographical references.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-12T11:44:31Z No. of bitstreams: 1 texto completo.pdf: 10511825 bytes, checksum: ff27fb15316ce502d8d27e94ab3c9b16 (MD5) / Made available in DSpace on 2017-04-12T11:44:31Z (GMT). No. of bitstreams: 1 texto completo.pdf: 10511825 bytes, checksum: ff27fb15316ce502d8d27e94ab3c9b16 (MD5) Previous issue date: 1997-03-25 / A produção de etanol a partir de soro de queijo suplementado com LB ou 0,5% de extrato de Ievedura por Klebsiella oxytoca M5A1, recombinante P2 (Kb.P2), em valores de pH controlado em 6,0; 6,5; 6,9; 7,0; e 7,3, apresentou baixo rendimento, o que indica ineficiência de estirpe Kb.P2 em utilizar a lactose do soro como substrato. A temperatura ótima para produção de enzima β-D-galactosidade foi 30°C. O pH e a temperatura de atividade ótima da enzima β-D-galactosidade foram 7,0 e 45°C, respectivamente. A enzima não apresentou estabilidade em temperatura superior a 35°C. Houve repressão catabólica de enzima, com a adição de glicose 10 minutos após a adição do indutor lPTG. A galactose adicionada em uma cultura paralela, no mesmo tempo, inibiu ligeiramente a produção de enzima. A β-D-galactosidase de Kb.P2 apresentou 37% de atividade em relação à E. coli K12, quando induzida com IPTG, indicando que Kb.P2 possui uma baixa expressão de enzima. As células rompidas com a prensa francesa ou as células permeabilizadas com tolueno apresentaram 1,3 vez mais atividade de β-D-galactosidase 30 minutos após a adição do indutor, comparado a células induzidas e não tratadas, o que indica baixa eficiência na entrada do substrato nas células. Os resultados permitem evidenciar que a fermentação de soro de queijo por Kb.P2 depende, ainda, do conhecimento e da manipulação do sistema da bactéria, visando utilizar lactose. / The fermentation of cheese whey, supplemented with LB or 0.5% yeast extract, by ethanologenic Klebsiella oxyfoca M5A1, recombinant P2 (Kb.P2), in media with the pH controlled at 6.0; 6.5; 6.9; 7.0. and 7.3, resulted in low ethanol yield. These results indicated that Kb.P2 was not expressing the Lac- operon efficiently. The optimum temperature for β-D-galactosidase production was 30°C. The best pH and temperature for β-D-galactosidase activity were 7.0 and 45°C, respectively. However, enzyme stability was reduced at temperatures higher than 35°C. The addition of glucose repressed β-D-galactosidase more than the addition of galactose. When compared to Escherichia coli K12. B-D-galactosidase induction with IPTG in Kb.P2 corresponded to only 37% of the activity observed for K12. This result showed that the expression of Lac-operon genes was weak. Cultures induced for 30 min with IPTG, and, then, treated in a French press or with toluene had 1.3 times more β-D-galactosidase activity than induced cells that were not ruptured, showing inefficient transport of the substrate into the cells. The possibility of Kb.P2 use for cheese whey fermentation depends on a better understanding of Lac-operon.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-06-23T11:41:54Z No. of bitstreams: 1 texto completo.pdf: 388479 bytes, checksum: 3f6213e3987bc7cbece94deab1128175 (MD5) / Made available in DSpace on 2017-06-23T11:41:54Z (GMT). No. of bitstreams: 1 texto completo.pdf: 388479 bytes, checksum: 3f6213e3987bc7cbece94deab1128175 (MD5) Previous issue date: 2001-03-15 / 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

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Ciência dos Alimentos, Florianópolis, 2014. / Made available in DSpace on 2015-02-05T20:40:56Z (GMT). No. of bitstreams: 1 330022.pdf: 5522183 bytes, checksum: 0735aaf23ec57931e46e27aa4526fa68 (MD5) Previous issue date: 2014 / O sorvete é um sistema coloidal complexo composto por uma emulsão constituída de gotículas de gordura, proteínas, bolhas de ar e de cristais dispersos em uma fase aquosa. O produto desejado deve ter alto overrun (aeração), textura macia, baixa taxa de derretimento e poucos cristais e açúcar, obtido pela aplicação das enzimas, uma alternativa inovadora e funcional na fabricação de sorvetes. Nesta pesquisa objetivou-se otimizaras condições operacionais da produção de sorvete diet de morango pela adição de ingredientes (concentrado proteico de soro de leite e edulcorantes) e das enzimas lactase e transglutaminase, utilizando a metodologia de superfície de resposta para avaliar seus efeitos sob diferentes parâmetros físico-químicos (overrun, textura, taxa de derretimento e formação de cristais) e químicos (lactose e proteínas) na formulação do sorvete. A enzima lactase ß-galactosidase (EC. 3.2.1.23) é a responsável pela hidrólise de ligações ß-galactosídicas da lactose, resultando em sua redução pela conversão em glicose e galactose. Já a enzima transglutaminase (EC 2.3.2.13) apresenta a capacidade de catalisar reações de transferência de grupos acil, formando ligações cruzadas (intra e intermoleculares) entre proteínas, peptídeos e aminas primárias, principalmente ligações covalentes entre resíduos de glutamina e lisina (ligações G-L), aumentando a fração das proteínas de alta massa molar. Portanto, o objetivo deste trabalho foi avaliar pelo planejamento experimental central composto as condições ideais de temperatura e concentração de transglutaminase e lactase, visando a uma formulação de sorvete diet com características físico-químicas adequadas, considerando ainda as propriedades microbiológicas e sensoriais. Para tanto, foram preparadas 18 formulações de sorvetes por processo descontínuo empregando diferentes concentrações das enzimas e temperaturas de incubação, segundo delineamento fatorial 23, com quatro repetições no ponto central, para averiguar o efeito das enzimas na qualidade do sorvete. Foram realizadas determinações de: overrun, textura, índice de derretimento, reação de polimerização das proteínas do soro de leite pela formação de bandas de proteinas, hidrólise da lactose e enumerações de cristais. Nas análises estatísticas dos resultados das superfícies de contorno empregou-se a análise de variância (ANOVA), seguida do teste de Tukey em 5% de probabilidade para todos os tratamentos. A formação de cristais nos sorvetes foi verificada utilizando ANOVA unicaldal, seguida do teste de Kruskal-Wallis (não paramétrico) e do pós-teste de Dunn comparando todos os tratamentos, em nível de significância de 5%. O planejamento experimental e a análise de desejabilidade auxiliaram-na escolha do tratamento 2 como ideal, empregando as enzimas lactase (0,4 g L-1) e transglutaminase (2,0 U g-1 proteína) a 40 oC. Este foi o tratamento que apresentou melhor textura; alto overrun; menor índice de derretimento; formação de bandas de eletroforese de proteínas de alta massa molar evidenciando a formação de polímeros por ligações cruzadas pela ação da enzima transglutaminase; e a atuação mais eficiente da enzima lactase, avaliada pela hidrólise da lactose por determinação cromatográfica, confirmados pela formação de pequenos cristais e açúcar, observados pela micrografia, contribuindo para uma textura mais lisa. Pela análise sensorial, o sorvete elaborado em tais condições foi o que obteve maior aceitabilidade entre os provadores não treinados. Dessa forma, a adição das enzimas lactase e transglutaminase produziu melhora nas propriedades de textura, pelo aumento da cremosidade e suavidade, produzindo um alimento funcional com maior digestibilidade e menos calorias.<br> / Abstract : Ice cream is a complex colloidal system comprised of an emulsion consisting of fat droplets, proteins, air bubbles and crystals dispersed in an aqueous phase. The desired product should have a high overrun (degree of aeration), soft texture, slow melting rate and a low amount of crystals and sugar, obtained through the application of enzymes, an innovative and functional alternative used in the production of ice creams. The aim of this research was to optimize the operational conditions for the production of diet strawberry ice cream through the addition of ingredients (milk whey protein concentrate and sweeteners) and enzymes (lactase and transglutaminase), using the response surface methodology to evaluate their effects on different physico-chemical (overrun, texture, melting rate and formation of crystals) and chemical (lactose and proteins) parameters of the ice cream formulation. The enzyme lactase (ß-galactosidase (EC 3.2.1.23) is responsible for the hydrolysis of lactose ß-galactoside bonds, resulting in their reduction through conversion to glucose and galactose. The enzyme transglutaminase (EC 2.3.2.13) is able to catalyze acyl transfer reactions, forming cross-links (intra and intermolecular) between proteins, peptides and primary amines, mainly covalent bonds between residues of glutamine and lysine (G-L bonds), increasing the fraction of proteins of high molecular mass. However, the objective of this research was to evaluate, through a central composite experimental design, the ideal conditions of temperature and transglutaminase and lactase concentrations, aimed at obtaining a diet ice cream formulation with suitable physico-chemical characteristics, considering also the microbiological and sensory properties. A total of 18 ice cream formulations were prepared by a discontinuous process employing different concentrations of enzymes and incubation temperatures, according to a 23 factorial design, with four repetitions at the central point, to investigate the effect of the enzymes on the ice cream quality. Analysis was carried out to determine the overrun, texture, melting index, milk whey protein polymerization reaction through the formation of protein bands, lactose hydrolysis and number of crystals. In the statistical analysis of the results for the response surfaces, analysis of variance (ANOVA) followed by the Tukey test (5% probability) was used for all treatments. The formation of crystals in the ice cream samples was verified using one-tailed ANOVA followed by the Kruskal-Wallis test (non-parametric) and the Dunn s post-hoc test comparing all treatments, at the 5% significance level. The experimental design and the desirability analysis aided the selection of the ideal treatment (treatment 2), in which the enzymes lactase (0.4 g L-1) and transglutaminase (2.0 U g-1 protein) and a temperature of 40 oC were employed. This was the treatment which provided the best texture, high overrun, the lowest melting index, formation of electrophoresis bands of proteins of high molecular mass evidencing the formation of polymers through cross-links due to the action of the enzyme transglutaminase and the most efficient action of the enzyme lactase, evaluated through the lactose hydrolysis determined by chromatography, verified by the formation of small crystals and sugar, observed on themicrograph, contributing to a smoother texture. The sensory analysis indicated that the ice cream prepared under these conditions had the greatest acceptability according to the untrained tasters. Thus, the addition of the enzymes lactase and transglutaminase improved the textural properties by increasing the creaminess and smoothness, producing a functional food with greater digestibility and reduced calories.

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. / Submitted by demia Maia (demiamlm@gmail.com) on 2016-05-19T13:46:24Z No. of bitstreams: 1 2012_tese_aflima.pdf: 2295796 bytes, checksum: d8694cc32f36b9bee8f170aa68a677be (MD5) / Approved for entry into archive by demia Maia (demiamlm@gmail.com) on 2016-05-19T13:47:54Z (GMT) No. of bitstreams: 1 2012_tese_aflima.pdf: 2295796 bytes, checksum: d8694cc32f36b9bee8f170aa68a677be (MD5) / Made available in DSpace on 2016-05-19T13:47:54Z (GMT). No. of bitstreams: 1 2012_tese_aflima.pdf: 2295796 bytes, checksum: d8694cc32f36b9bee8f170aa68a677be (MD5) Previous issue date: 2012 / 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.

Made available in DSpace on 2014-06-11T19:35:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2005Bitstream added on 2014-06-13T18:46:07Z : No. of bitstreams: 1 adalberto_pr_dr_araiq.pdf: 786247 bytes, checksum: cf55691544529062c4d2288dd8709c7a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / 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.