In this study, we evaluated the potential of water-in-oil microemulsions for the separation of beta-galactosidase (lactase) from other proteins. The ability of beta-galactosidase to break down the milk carbohydrate lactose gives the enzyme considerable commercial importance. The extent of solubilization of a commercial Kluyveromyces lactis preparation of beta-galactosidase into microemulsion droplets formed from 200 mM bis (2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane was measured as a function of buffer type, pH, ionic strength, and protein concentration. Our results showed that, due to the large molecular weight of beta-galactosidase (MW~ 220-240 kDa, dimeric form), the enzyme was taken up by the microemulsion droplets mainly under very low salt conditions. Based on these results, we designed a one-step separation procedure, in which a small volume of aqueous buffer containing the protein mixture is added to an organic surfactant solution. Microemulsion droplets form in the oil and capture protein impurities of smaller molecular weights, while excluding the high molecular weight target protein. This causes the beta-galactosidase to be expelled into a newly formed aqueous phase. The feasibility of this one-step process as a bioseparation tool was demonstrated on a feed consisting of an equal mixture of beta-galactosidase and the test protein beta-lactoglobulin. Recovery and separation of the two proteins was analyzed as function of buffer type, pH, ionic strength, and protein concentration. Results showed that separation was most complete at 100 mM KCl salt concentration, where the droplets were big enough to carry beta-lactoglobulin but too small for lactase. At 100 mM salt concentration, we recovered 92% of the total lactase activity in a virtually pure form. The same separation scheme was then tested on crude extract obtained from a cell culture broth of the yeast Kluyveromyces lactis. Cells of the yeast K. lactis were disrupted by minibeadbeater, forming a crude extract that was used as the feed in our separation process. A 5.4-fold purification factor of the extract was achieved, with 96% activity recovery. The results showed our one-step separation process to be an interesting method for the production of beta-galactosidase as a technical enzyme: it has the potential to achieve a continuous, large-scale partial purification of the enzyme, potentially reducing the number of steps required in downstream process.

No description available.

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.

This research explores Isothermal Titration Calorimetry as a method for measuring beta-galactosidase activity directly and continuously in milk, sweet whey, sweet whey permeate, acid whey, and acid whey permeate. Beta-galactosidase in various concentrations was injected into each of the liquid dairy products spiked with lactose to verify if the heat rate from the enzymatic reaction could be observed. In addition, a consistent concentration of beta-galactosidase was injected into various concentrations of lactose in the products, to observe the heat rates from the enzymatic reaction. There was exothermic activity that never returned to baseline demonstrated in milk, sweet whey, and sweet whey permeate with beta-galactosidase from Kluyveromyces lactis in runs done in the isothermal titration calorimeter. The baseline was approximately 3-9 uJ/s above the control's baseline at the end of the runs. The exothermic activity ranged from approximately 2-10 uJ/s and did not return to baseline when beta-galactosidase concentrations were varied and lactose concentrations remained the same. The exothermic heat rate was approximately 3-7 uJ/s when lactose concentrations were varied and enzyme concentrations remained the same. With runs with increasing lactose concentrations, there was no corresponding increase in the exothermal reaction indicating saturation of the enzyme. There was a short exothermic reaction(s), ranging from approximately 3-26 uJ/s, demonstrated when varying concentrations of beta-galactosidase from Aspergillus oryzae in acid whey and acid whey permeate were injected into a consistent concentration of lactose in acid whey and acid whey permeate. There was a pattern of increasing heat with increasing concentrations of enzyme, with some of these differences being statistically significant. There was also a short exothermic reaction(s), ranging from approximately 2-17 uJ/s, demonstrated when a consistent concentration of beta-galactosidase from Aspergillus oryzae was injected into varying concentrations of lactose. There was a pattern of increasing heat rate with increasing concentrations of lactose, with some of these differences being statistically significant. This research demonstrates that ITC is a useful method for measuring residual beta-galactosidase and/or residual lactose in liquid dairy products. This research leads to further understanding of how enzymes and substrates interact directly in the food matrix, rather than in an isolated environment.

No description available.

Pichia pastoris is a methylotrophic yeast that has been used in both research and industrial settings for recombinant protein expression due to the ease of genetically modifying its genome, its ability to grow to large densities in inexpensive media, and its capability to perform posttranslational modifications. Multiple tools such as the cis -acting factors MATα secretion signal and MBP fusion partners, and trans-acting modifications such as the bgs mutants have increased heterologous protein secretion. Although these techniques have already been used, their effects on the protein secretory pathway have yet to be elucidated. In this study, fluorescence microscopy was used to compare the localization of proteins expressed with the mutated MATα with the deletion of amino acids 57-70 to the wild type MATα secretion signal. Additional fluorescence microscopy was completed to visualize the localization of MBP-EGFP and EGFP-MBP fusion proteins and their spatial relativity to organelle markers. EGFP-MBP was used to further distinguish the properties of multiple bgs mutants. Additionally, secreted lipase activity levels were evaluated in bgs13 strains expressing either the wild type or the mutated MAT&agr; signal peptide. The results indicated that regardless of their differences, the MATα secretion signals and bgs mutants transported their cargo proteins through similar pathways within the cells. The results of the MBP fusion proteins suggest that the arrangement of MBP significantly influences protein secretion and localization. Lastly, the bgs13 mutant with MATα secretion signals demonstrated that lipase activity increased additively when cis- and trans -acting mutations were combined. Ultimately, these results can provide better understanding of each modified factor and the protein sorting pathway, leading to potential techniques that optimize protein secretion in P. pastoris .

Many consumers are unable to enjoy the benefits of milk, due to lactose-intolerance. Lactose-free milk is available, but at about 2 times the cost of regular milk or greater, it may be difficult for consumers to afford. The high cost of lactose-free milk is in part due to the added cost of the lactose hydrolysis process. Hydrolysis at refrigerated temperatures, possibly in the bulk tank or package, could increase the flexibility of the process, and potentially reduce the cost. A rapid β-galactosidase assay was used to determine the relative activity of commercially available lactase samples at different temperatures. Four enzymes exhibited low-temperature activity and were added to refrigerated raw and pasteurized milk at various concentrations and allowed to react for various lengths of time. The degree of lactose hydrolysis by each of the enzymes as a function of time and enzyme concentration was determined by HPLC. The two most active enzymes, as determined by the β-galactosidase assay, hydrolyzed over 98% of the lactose in 24 hours at 2°C using the supplier recommended dosage. The other two enzymes hydrolyzed over 95% of the lactose in 24 hours at two times the supplier recommended dosage at 2°C. Results were consistent in all milk types tested. The results show that it is feasible to hydrolyze lactose during refrigerated storage of milk using currently available enzymes.