RESUMO
Yeast's beta-galactosidase is an intracellular enzyme, through which it is possible to determine in vivo its activity as a biocatalyst in the lactose hydrolysis. Permeabilization process was used for transforming the microorganisms cells into biocatalysts with an enhanced enzyme activity. The potential application of this enzyme technology in industrial process depends mainly on the enzyme activity. Beta-galactosidase enzyme that hydrolyzes lactose, for instance, is largely dependent on the reaction time and its stability under different physical conditions, such as pH, temperature and enzyme concentration. The objective of this study was to optimize the cellular permeabilization process of Kluyveromyces marxianusCCT 3172 and Saccharomyces fragilisCCT 7586 cultured in cheese whey for lactose hydrolysis. Box-Behnken design was carried out for cell permeabilization with three independent variables, ethanol concentration, permeabilization time and temperature. The best permeability conditions for K. marxianusCCT 3172 were 27% (v v-1) ethanol, 3 min at 20ºC, with specific enzymatic activity of 0.98 U mg-1.For S. fragilisCCT 7586, a specific enzymatic activity of 1.31 U mg-1was achieved using 45% (v v-1) of ethanol, 17 min. of reaction under 17ºC. Thus, it was concluded that cellular permeabilization with ethanolis an efficient process to determine beta-galactosidase activity.(AU)
Assuntos
Permeabilidade , Kluyveromyces , beta-Galactosidase , Soro do Leite , Lactose , Leveduras , Queijo , Enzimas/biossínteseRESUMO
RESUMEN La gangliosidosis GM1 es un trastorno lisosomal caracterizada por la acumulación de gangliósido GM1 (glucoesfingolípido) en el sistema nervioso central (SNC) y visceral, debido a la deficiencia de la enzima beta-galactosidase (hidrolasa lisosomal). Afecta principalmente al SNC y las vísceras y produce importantes anomalías esqueléticas, que a menudo ocurren con la presencia de linfocitos vacuolados en la muestra de la sangre periférica o médula ósea. Tiene tres formas de presentación, lo que dificulta aún más su identificación debido al amplio espectro clínico. El presente estudio tiene como objetivo describir un caso de gangliosidosis GM1 en un paciente masculino, nacido a las 38 semanas. Hasta el momento, no existe un tratamiento efectivo para la gangliosidosis GM1, es decir, el portador de la enfermedad solo recibe medidas sintomáticas y paliativas. Por tanto, el diagnóstico precoz de la enfermedad es de suma importancia, ya que su única forma de prevención, actualmente, es a través del consejo genético.
RESUMO
Here, we report the effect of polyethylene glycol (PEG6000)-induced molecular crowding (MC) on the catalytic activity and thermal stability of Kluyveromyces lactis ß-galactosidase (ß-Gal). The ß-Gal-catalyzed hydrolysis of o-nitrophenyl-ß-d-galactopyranoside followed a Michaelian kinetics at [PEG6000] ≤ 25% w/v and positive cooperativity at higher concentrations (35% w/v PEG6000). Compared with dilute solutions, in the MC media, ß-Gal exhibited stronger thermal stability, as shown by the increase in the residual activity recovered after preincubation at high temperatures (e.g., 45 °C) and by the slower inactivation kinetics. Considering the effects of water thermodynamic activity on the reaction kinetics and protein structure and the effect of the exclusion volume on protein conformation, we suggest that changes in the protein oligomerization state and hydration could be the responsible for the behavior observed at the highest MC levels assayed. These results could be relevant and should be taken into account in industrial food processes applying ß-Gal from K. lactis.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Kluyveromyces/enzimologia , beta-Galactosidase/química , beta-Galactosidase/metabolismo , Biocatálise , Estabilidade Enzimática , Temperatura Alta , Cinética , Kluyveromyces/química , Polietilenoglicóis/químicaRESUMO
Se evaluó el efecto de la temperatura sobre la desnaturalización de proteínas y la reacción de Maillard en leche entera y descremada con lactosa hidrolizada. Las leches hidrolizadas se trataron térmicamente a 100, 110, 120 y 130 °C durante un período de 1 hora y se midió la concentración de glucosa, el grado de pardeamiento y la desnaturalización de proteínas. El grado de dorado en la leche entera varió de 14.4 (100 °C) a 42.6 (130 °C). Para la leche descremada fue de 20.2 (100 °C) a 38.0 (130 °C). La concentración de glucosa en leche entera (47% p/v) y en leche descremada (41% p/v) después del tratamiento térmico (130 °C) mostró una reducción significativa en relación con el control (25 °C). El efecto de la temperatura en la desnaturalización de proteínas en leche entera y descremada en relación con el control (25 °C) fue del 100%. La leche tratada térmicamente con lactosa hidrolizada promovió la desnaturalización de proteínas con un aumento del pardeamiento característico de la reacción de Maillard, lo que afectó la calidad nutricional.
The effect of temperature in protein denaturation and Maillard reaction in whole and skim milk with hydrolyzed lactose was evaluated. Hydrolyzed milk was thermally treated at 100, 110, 120 and 130 °C over a period of 1 hour and glucose concentration, browning degree and protein denaturation were measured. The browning degree in whole milk varied from 14.42 (100 °C) to 42.63 (130 °C) and 20.21 (100 °C) to 38.03 (130 °C) in skim milk. Glucose concentration in whole milk (47% - w/v) and skim milk (41% - w/v) after heat treatment (130 °C) showed a significant reduction in relation to the control (25 °C). The temperature effect in protein denaturation in whole and skim milk in relation to the control (25 °C) was 100%. Thermally treated milk with hydrolyzed lactose promoted protein denaturation with increasing browning characteristic of the Maillard reaction, thus affecting the nutritional quality.
Assuntos
Desnaturação Proteica , Temperatura , Reação de Maillard , Leite/química , Lactose/química , Tratamento Térmico , beta-Galactosidase , Cor , Glucose/análise , HidróliseRESUMO
OBJECTIVE: To evaluate the clinical presentation of patients with GM1 gangliosidosis and to determine whether specific clinical or biochemical signs could lead to a prompt diagnosis. STUDY DESIGN: We retrospectively analyzed clinical, biochemical, and genetic data of 22 patients with GM1 gangliosidosis from 5 metabolic centers in Germany and Austria. RESULTS: Eight patients were classified as infantile, 11 as late-infantile, and 3 as juvenile form. Delay of diagnosis was 6 ± 2.6 months in the infantile, 2.6 ± 3.79 years in the late-infantile, and 14 ± 3.48 years in the juvenile form. Coarse facial features, cherry red spots, and visceromegaly occurred only in patients with the infantile form. Patients with the late-infantile and juvenile forms presented with variable neurologic symptoms. Seventeen patients presented with dystonia and 14 with dysphagia. Laboratory analysis revealed an increased ASAT concentration (13/20), chitotriosidase activity (12/15), and pathologic urinary oligosaccharides (10/19). Genotype analyses revealed 23 causative or likely causative mutations in 19 patients, 7 of them being novel variants. In the majority, a clear genotype-phenotype correlation was found. CONCLUSIONS: Diagnosis of GM1 gangliosidosis often is delayed, especially in patients with milder forms of the disease. GM1 gangliosidosis should be considered in patients with progressive neurodegeneration and spastic-dystonic movement disorders, even in the absence of visceral symptoms or cherry red spots. ASAT serum concentrations and chitotriosidase activity may be of value in screening for GM1 gangliosidosis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , DNA/genética , Gangliosidose GM1/genética , Mutação , beta-Galactosidase/genética , Adolescente , Áustria/epidemiologia , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Seguimentos , Gangliosidose GM1/diagnóstico , Gangliosidose GM1/epidemiologia , Genótipo , Alemanha/epidemiologia , Humanos , Incidência , Lactente , Masculino , Fenótipo , Estudos Retrospectivos , Adulto Jovem , beta-Galactosidase/metabolismoRESUMO
GM1 gangliosidosis is a lysosomal storage disorder caused by ß-galactosidase deficiency. To date, prospective studies for GM1 gangliosidosis are not available, and only a few have focused on the adult form. This retrospective cross-sectional study focused on clinical findings in Brazilian patients with the adult form of GM1 gangliosidosis collected over 2 years. Ten subjects were included in the study. Eight were males and two females, with median age at diagnosis of 11.5 years (IQR, 4-34 years). Short stature and weight below normal were seen in five out of the six patients with data available. Radiological findings revealed that the most frequent skeletal abnormalities were beaked vertebrae, followed by hip dysplasia, and platyspondyly. Neurological examination revealed that dystonia and swallowing problems were the most frequently reported. None of the patients presented hyperkinesia, truncal hypertonia, Parkinsonism, or spinal cord compression. Clinical evaluation revealed impairment in activities of cognitive/intellectual development and behavioral/psychiatric disorders in all nine subjects with data available. Language/speech impairment (dysarthria) was found in 8/9 patients, fine motor and gross motor impairments were reported in 7/9 and 5/9 patients, respectively. Impairment of cognition and daily life activities were seen in 7/9 individuals. Our findings failed to clearly identify typical early or late alterations presented in GM1 gangliosidosis patients, which confirms that it is a very heterogeneous condition with wide phenotypic variability. This should be taken into account in the evaluation of future therapies for this challenging condition.
RESUMO
La galactosialidosis (OMIM #256540) es una enfermedad metabólica lisosomal causada por mutaciones en el gen CTSA, que codifica la proteína protectora catepsina A. La pérdida de función de dicha proteína causa, secundariamente, un déficit combinado de dos enzimas, beta-galactosidasa y neuraminidasa. Se expone el caso de un paciente que presentó manifestaciones clínicas compatibles con el subtipo infantil tardío de galactosialidosis. El análisis bioquímico mostró déficits de las dos enzimas implicadas, mientras que el estudio molecular reveló dos mutaciones: una nueva mutación nunca antes descrita, p.His475Pro (c.1424 A>C), y una mutación previamente reportada, p.Arg441Cys (c.1321C>T), localizadas en los exones 15 y 14, respectivamente.
Galactosialidosis (OMIM #256540) is an autosomal recessive lysosomal storage disorder caused by mutations in the CTSAgene, which encodes the protective protein cathepsin A. The loss of function of this protein causes a secondarily deficiency of beta-galactosidase and N-acetyl-α-neuraminidase enzymes activities. We describe the clinical, biochemical and molecular analysis of a case report with a phenotype compatible with the late infantile form. The biochemical analysis reveled deficiencies of beta-galactosidase and neuraminidase activities in dried blood spot and fibroblasts and the molecular study showed two missense mutations in the CTSA gene: a previously reported mutation, p.Arg441Cys (c.1321C>T), and a novel mutation, p.His475Pro (c.1424 A>C), located in exons 14 and 15, respectively.
Assuntos
Humanos , Masculino , Pré-Escolar , Doenças por Armazenamento dos Lisossomos/genética , Catepsina A/genética , Mutação , Doenças por Armazenamento dos Lisossomos/diagnósticoRESUMO
Galactosialidosis (OMIM #256540) is an autosomal recessive lysosomal storage disorder caused by mutations in the CTSA gene, which encodes the protective protein cathepsin A. The loss of function of this protein causes a secondarily deficiency of beta-galactosidase and N-acetyl-a-neuraminidase enzymes activities. We describe the clinical, biochemical and molecular analysis of a case report with a phenotype compatible with the late infantile form. The biochemical analysis reveled deficiencies of beta-galactosidase and neuraminidase activities in dried blood spot and fibroblasts and the molecular study showed two missense mutations in the CTSA gene: a previously reported mutation, p.Arg441Cys (c.1321C>T), and a novel mutation, p.His475Pro (c.1424 A>C), located in exons 14 and 15, respectively.
La galactosialidosis (OMIM #256540) es una enfermedad metabólica lisosomal causada por mutaciones en el gen CTSA, que codifica la proteina protectora catepsina A. La pérdida de función de dicha proteína causa, secundariamente, un déficit combinado de dos enzimas, beta-galactosidasa y neuraminidasa. Se expone el caso de un paciente que presentó manifestaciones clínicas compatibles con el subtipo infantil tardío de galactosialidosis. El análisis bioquímico mostró déficits de las dos enzimas implicadas, mientras que el estudio molecular reveló dos mutaciones: una nueva mutación nunca antes descrita, p.His475Pro (c.1424 A>C), y una mutación previamente reportada, p.Arg441Cys (c.1321C>T), localizadas en los exones 15 y 14, respectivamente.
Assuntos
Catepsina A/genética , Doenças por Armazenamento dos Lisossomos/genética , Mutação , Pré-Escolar , Humanos , Doenças por Armazenamento dos Lisossomos/diagnóstico , MasculinoRESUMO
BACKGROUND: Lactose intolerance is characterized by the absence of the enzyme lactase (beta-galactosidase) and affects two thirds of the world adult population. Our aim was to evaluate a lactase gastro-resistant formulation to ensure increased activity in the action site of the enzyme (lumen of the small intestine). Simultaneously, we also evaluated the commercial product stability and enzyme activity, because the product containing beta-galactosidase is classified as food supplement according to the Food and Drug Administration (FDA), so it is free to pass quality testing, efficacy and stability. So, it is possible that contain some irregularities as to the content and enzymatic activity. METHODS: The dissolution assay was performed using a dissolution test system and commercial product and the gastro-resistant formulation were evaluated according to a method adapted to the conditions recommended by United States Pharmacopeia (US Pharmacopeia) for gastro-resistant formulations. For the assessment of enzymatic activity throughout the dissolution test was employed the official method of lactase assay described in US Pharmacopoeia. This method is based on a colorimetric reaction which the substrate reacts with the enzyme generate a colored product further analyzed by UVVisible spectrophotometry. RESULTS: When carrying out dissolution test in commercial product it is noted that the existing formulation is not able to protect the enzyme from degrading action of gastric environment (a loss of 86.0 ± 0.8% of lactase activity was observed). Our proposed gastro-resistant pharmaceutical form there was no loss of activity during the acid step and the end of the dissolution test the found activity was 95 ± 1.3%. CONCLUSION: The formulations proposed in this work using hypromellose capsules ensure the enzymatic activity of lactase, preventing its contact with the acid medium. For the other side, the results of commercial tablets for lactase release indicate a significant loss of enzyme activity due to the immediate release of the enzyme in the simulated gastric fluids.
Assuntos
Lactase/química , Cápsulas/química , Química Farmacêutica/métodos , Solubilidade , Comprimidos/química , Estados Unidos , United States Food and Drug AdministrationRESUMO
ABSTRACT Background Primary hypolactasia is a common condition where a reduced lactase activity in the intestinal mucosa is present. The presence of abdominal symptoms due to poor absorption of lactose, which are present in some cases, is a characteristic of lactose intolerance. , Objective Evaluate the efficacy of a product containing exogenous lactase in tablet form compared to a reference product with proven effectiveness in patients with lactose intolerance. Methods Multicentre, randomized, parallel group, single-blind, comparative non-inferiority study. One hundred twenty-nine (129) adult lactose intolerance patients with hydrogen breath test results consistent with a diagnosis of hypolactasia were randomly assigned to receive the experimental product (Perlatte(r) - Eurofarma Laboratórios S.A.) or the reference product (Lactaid(r) - McNeilNutritionals, USA) orally (one tablet, three times per day) for 42 consecutive days. Results Data from 128 patients who actually received the studied treatments were analysed (66 were treated with the experimental product and 62 with the reference product). The two groups presented with similar baseline clinical and demographic data. Mean exhaled hydrogen concentration tested at 90 minutes after the last treatment (Day 42) was significantly lower in the experimental product treated group (17±18 ppm versus 34±47 ppm) in the per protocol population. The difference between the means of the two groups was -17 ppm (95% confidence interval [95% CI]: -31.03; -3.17). The upper limit of the 95% CI did not exceed the a priori non-inferiority limit (7.5 ppm). Secondary efficacy analyses confirmed that the treatments were similar (per protocol and intention to treat population). The tolerability was excellent in both groups, and there were no reports of serious adverse events related to the study treatment. Conclusion The experimental product was non-inferior to the reference product, indicating that it was an effective replacement therapy for endogenous lactase in lactose intolerance patients.
RESUMO Contexto A hipolactasia primária é uma condição muito frequente na qual há redução da atividade da lactase na mucosa intestinal.A presença de sintomas abdominais devidos à má absorção da lactose presente em alguns casos caracteriza a intolerância à lactose. Objetivo Avaliar a eficácia de um produto contendo lactase exógena em comprimidos comparativamente a de um produto comparador com eficácia comprovada em pacientes portadores de intolerância à lactose. Métodos Estudo multicêntrico, randomizado, de grupos paralelos, com investigador cego, comparativo de não-inferioridade. Cento e vinte e nove (129) pacientes adultos portadores de intolerância à lactose e teste do hidrogênio no ar expirado compatível com o diagnóstico de hipolactasia foram randomizados para receber o produto experimental (Perlatte(r) - Eurofarma Laboratórios S.A.) ou o produto comparador (Lactaid(r) - McNeil Nutritionals, EUA), por via oral (um comprimido, três vezes ao dia), durante 42 dias consecutivos. Resultados Os dados dos 128 pacientes que efetivamente receberam o tratamento do estudo foram avaliados (66 tratados com o produto experimental e 62 com o produto comparador). Os dois grupos se mostraram homogêneos quanto aos dados demográficos e clínicos basais. A média da concentração do hidrogênio expirado aos 90 minutos no teste realizado ao final do tratamento (Dia 42) foi significativamente menor no grupo tratado com o produto experimental (17±18 ppm versus 34±47 ppm na população por protocolo). A diferença entre as médias dos dois grupos foi de -17 ppm (intervalo de confiança de 95% [IC95%]: -31,03; -3,17). O limite superior do IC95% não ultrapassou a margem de não-inferioridade estipulada a priori (7,5 ppm). As análises secundárias de eficácia confirmaram a semelhança entre os tratamentos (populações por protocolo e com intenção de tratamento). A tolerabilidade foi excelente em ambos os grupos e não houve relato de eventos adversos graves relacionados ao produto. Conclusão O produto experimental se mostrou não-inferior ao produto comparador, indicando sua eficácia no tratamento substitutivo da lactase endógena em pacientes portadores de intolerância à lactose.
Assuntos
Humanos , Masculino , Feminino , Adolescente , Adulto , Adulto Jovem , Lactase/administração & dosagem , Lactase/deficiência , Intolerância à Lactose/tratamento farmacológico , Método Simples-Cego , Administração Oral , Resultado do Tratamento , Hidrogênio/análise , Lactose/metabolismo , Intolerância à Lactose/diagnóstico , Pessoa de Meia-IdadeRESUMO
In a search for better comprehension of ß-galactosidase function and specificity, we solved the crystal structures of the GH42 ß-galactosidase BbgII from Bifidobacterium bifidum S17, a well-adapted probiotic microorganism from the human digestive tract, and its complex with d-α-galactose. BbgII is a three-domain molecule that forms barrel-shaped trimers in solution. BbgII interactions with d-α-galactose, a competitive inhibitor, showed a number of residues that are involved in the coordination of ligands. A combination of site-directed mutagenesis of these amino acid residues with enzymatic activity measurements confirmed that Glu161 and Glu320 are fundamental for catalysis and their substitution by alanines led to catalytically inactive mutants. Mutation Asn160Ala resulted in a two orders of magnitude decrease of the enzyme kcat without significant modification in its Km , whereas mutations Tyr289Phe and His371Phe simultaneously decreased kcat and increased Km values. Enzymatic activity of Glu368Ala mutant was too low to be detected. Our docking and molecular dynamics simulations showed that the enzyme recognizes and tightly binds substrates with ß1â6 and ß1â3 bonds, while binding of the substrates with ß1â4 linkages is less favorable. DATABASE: Structural data are available in the PDB under the accession numbers 4UZS and 4UCF.
Assuntos
Proteínas de Bactérias/metabolismo , Bifidobacterium bifidum/enzimologia , Galactose/metabolismo , Galactosidases/metabolismo , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bifidobacterium bifidum/genética , Sítios de Ligação/genética , Biocatálise/efeitos dos fármacos , Domínio Catalítico , Cristalografia por Raios X , Galactose/química , Galactose/farmacologia , Galactosidases/química , Galactosidases/genética , Cinética , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Domínios Proteicos , Multimerização Proteica , Especificidade por SubstratoRESUMO
One of the greatest challenges for dairy industries is the correct destination of all the whey generated during cheese making, considering its high impact, the large volume created, and its technological potential. Enzymatic hydrolysis of cheese whey lactose is a biotechnological alternative. However, one of the limiting factors of its use is the relatively high cost of the enzymes, which could be lowered with the immobilization of these biocatalysts. Considering this context, the objective of this research was to evaluate the commercial Kluyveromyces lactis -galactosidase enzyme immobilized in calcium alginate spheres and gelatin, using glutaraldehyde and concanavalin A (ConA) as modifying agents in the hydrolysis of cheese whey lactose process. Results have shown that the enzyme encapsulation complexed with ConA in alginate-gelatin spheres, without glutaraldehyde in the immobilization support, has significantly increased the hydrolysis of lactose rate, achieving a maximum conversion of 72%.(AU)
Um dos grandes desafios das indústrias de laticínios é destinar de forma correta todo o soro gerado durante a produção de queijo, devido ao seu impacto ambiental, grande volume gerado e potencial tecnológico. A hidrólise enzimática da lactose presente no soro de queijo é uma alternativa biotecnológica. Contudo, um dos fatores limitantes de sua utilização é o custo relativamente alto das enzimas, o que poderia ser minimizado com a imobilização destes biocatalisadores. Baseado nesse contexto, o objetivo do presente trabalho foi avaliar a enzima comercial -galactosidase de Kluyveromyces lactis , imobilizada em esferas de alginato de cálcio e gelatina, empregando o glutaraldeído e a concanavalina A (ConA) como agentes modificadores, no processo de hidrólise da lactose presente no soro de queijo. Os resultados obtidos demonstraram que o encapsulamento da enzima complexada com ConA em esferas de alginato-gelatina, sem a presença de glutaraldeído no meio de imobilização, aumentou de modo significativo o teor de hidrólise da lactose, obtendo conversão máxima de 72%.(AU)
Assuntos
Queijo/análise , Soro , Kluyveromyces , beta-Galactosidase , Lactose , HidróliseRESUMO
One of the greatest challenges for dairy industries is the correct destination of all the whey generated during cheese making, considering its high impact, the large volume created, and its technological potential. Enzymatic hydrolysis of cheese whey lactose is a biotechnological alternative. However, one of the limiting factors of its use is the relatively high cost of the enzymes, which could be lowered with the immobilization of these biocatalysts. Considering this context, the objective of this research was to evaluate the commercial Kluyveromyces lactis -galactosidase enzyme immobilized in calcium alginate spheres and gelatin, using glutaraldehyde and concanavalin A (ConA) as modifying agents in the hydrolysis of cheese whey lactose process. Results have shown that the enzyme encapsulation complexed with ConA in alginate-gelatin spheres, without glutaraldehyde in the immobilization support, has significantly increased the hydrolysis of lactose rate, achieving a maximum conversion of 72%.
Um dos grandes desafios das indústrias de laticínios é destinar de forma correta todo o soro gerado durante a produção de queijo, devido ao seu impacto ambiental, grande volume gerado e potencial tecnológico. A hidrólise enzimática da lactose presente no soro de queijo é uma alternativa biotecnológica. Contudo, um dos fatores limitantes de sua utilização é o custo relativamente alto das enzimas, o que poderia ser minimizado com a imobilização destes biocatalisadores. Baseado nesse contexto, o objetivo do presente trabalho foi avaliar a enzima comercial -galactosidase de Kluyveromyces lactis , imobilizada em esferas de alginato de cálcio e gelatina, empregando o glutaraldeído e a concanavalina A (ConA) como agentes modificadores, no processo de hidrólise da lactose presente no soro de queijo. Os resultados obtidos demonstraram que o encapsulamento da enzima complexada com ConA em esferas de alginato-gelatina, sem a presença de glutaraldeído no meio de imobilização, aumentou de modo significativo o teor de hidrólise da lactose, obtendo conversão máxima de 72%.
Assuntos
Hidrólise , Kluyveromyces , Lactose , Queijo/análise , Soro , beta-GalactosidaseRESUMO
The permeabilization was used to transform microorganisms in cell biocatalysts with high enzymatic activity. The Saccharomyces fragilis IZ 275 yeast cells were permeabilized with ethanol, as permeabilizing agent. To optimize the permeabilization conditions were used the design of Box-Behnken 15 trials (3 central points). The independent variables and their levels were ethanol (29, 32 and 35%), temperature (15, 20 and 25°C) and time (15, 20 and 25 min). The answer (Y) function has beta-galactosidase activity (U mg-1). The optimum conditions for obtaining a high enzymatic activity were observed in 35% ethanol concentration, temperature 15ºC and 20 min. treatment time. The maximum activity of the enzyme beta-galactosidase obtained was 10.59 U mg-1. The permeabilization of the S. fragilis IZ 275 cells was efficient.
A permeabilização foi usada para transformar células de microrganismos em biocatalisadores com alta atividade enzimática. As células de levedura de Saccharomyces fragilis IZ 275 foram permeabilizadas com etanol, como agente permeabilizante. Para otimizar as condições de permeabilização foi utilizado o delineamento de Box-Behnken com 15 ensaios (3 repetições no ponto central) . As variáveis independentes e seus níveis foram etanol (29, 32 e 35%), temperatura (15, 20 e 25ºC) e tempo (15, 20 e 25 min.). A função resposta (Y) foi atividade de beta-galactosidase (U mg-1). As condições ótimas para a obtenção de uma alta atividade enzimática foram observadas em 35% de concentração de etanol, temperatura de 15°C e tempo de tratamento de 20 minutos. A máxima atividade da enzima beta-galactosidase obtida foi de 10.59 U mg-1. A permeabilização das células de S. fragilis IZ 275 foi eficiente.
Assuntos
Saccharomyces , beta-Galactosidase , Permeabilidade , Saccharomyces , Leveduras , Biotecnologia , Biocatálise , Hidrólise , LactoseRESUMO
The permeabilization was used to transform microorganisms in cell biocatalysts with high enzymatic activity. The Saccharomyces fragilis IZ 275 yeast cells were permeabilized with ethanol, as permeabilizing agent. To optimize the permeabilization conditions were used the design of Box-Behnken 15 trials (3 central points). The independent variables and their levels were ethanol (29, 32 and 35%), temperature (15, 20 and 25°C) and time (15, 20 and 25 min). The answer (Y) function has betagalactosidase activity (U mg-1). The optimum conditions for obtaining a high enzymatic activity were observed in 35% ethanol concentration, temperature 15ºC and 20 min. treatment time. The maximum activity of the enzyme beta-galactosidase obtained was 10.59 U mg-1. The permeabilization of the S. fragilis IZ 275 cells was efficient.(AU)
A permeabilização foi usada para transformar células de microrganismos em biocatalisadores com alta atividade enzimática. As células de levedura de Saccharomyces fragilis IZ 275 foram permeabilizadas com etanol, como agente permeabilizante. Para otimizar as condições de permeabilização foi utilizado o delineamento de Box-Behnken com 15 ensaios (3 repetições no ponto central). As variáveis independentes e seus níveis foram etanol (29, 32 e 35%), temperatura (15, 20 e 25ºC) e tempo (15, 20 e 25 min.). A função resposta (Y) foi atividade de beta-galactosidase (U mg-1). As condições ótimas para a obtenção de uma alta atividade enzimática foram observadas em 35% de concentração de etanol, temperatura de 15°C e tempo de tratamento de 20 minutos. A máxima atividade da enzima beta-galactosidase obtida foi de 10.59 U mg-1. A permeabilização das células de S. fragilis IZ 275 foi eficiente.(AU)
Assuntos
Saccharomyces/citologia , Saccharomyces/genética , Hidrólise , BiotecnologiaRESUMO
OBJETIVO: O objetivo deste estudo foi determinar a prevalência e gravidade das complicações oculares em pacientes com mucopolissacaridoses (MPS). MÉTODOS: Vinte e nove pacientes com diagnóstico de mucopolissacaridoses foram estudados. Foram avaliados: idade, sexo, acuidade visual, presença de estrabismo, erros refrativos, exame de fundo de olho, pressão intraocular, espessura corneal central e ultrassonografia ocular. RESULTADOS: Foram avaliados três pacientes com MPS I (12 por cento), 11 pacientes com MPS II (37,9 por cento), um paciente com MPS III (3,4 por cento) e 14 pacientes com MPS VI (48,3 por cento). A média de idade foi de 9,5 anos (DP 5,5). Observou-se hipermetropia em 88,5 por cento (23 pacientes) e astigmatismo em 51,7 por cento (15 pacientes). A média da acuidade visual corrigida foi de 0,45 logMAR (DP 0,68). A média do equivalente esférico foi +3,57 D (DP 2,46) e da pressão intraocular foi 17 mmHg (DP 3,9). Os achados mais comuns foram: espessamento palpebral 24,1 por cento (7 pacientes); opacidade da córnea, 55,2 por cento dos casos (16 pacientes); atrofia do nervo óptico, 23,1 por cento (6 pacientes); dobras radiais na retina 24 por cento (7 pacientes). O fundo de olho não foi examinado em 3 pacientes devido à opacidade de córnea. A média da espessura do complexo esclera-retina-coroide (ERC) medida por ultrassom foi de 1,78 mm (DP 0,51). CONCLUSÃO: Os achados oftalmológicos mais proeminentes foram espessamento palpebral, diminuição da acuidade visual, hipermetropia moderada, opacidade da córnea, dobras radiais na retina perimacular e atrofia do nervo óptico.
PURPOSE: The objective of this study was to determine the prevalence and severity of ocular complications in patients with mucopolysaccharidosis (MPS). METHODS: Twenty-nine patients with diagnosis of mucopolysaccharidosis were studied. Age, gender, visual acuity, presence of strabismus, refractive error, fundus examination, intraocular pressure, central corneal thickness and ocular echography were assessed for each individual. RESULTS: There were three patients with MPS I (12 percent), eleven patients with MPS II (37.9 percent), one patient with MPS III (3.4 percent) and fourteen patients with MPS VI (48.3 percent). Mean age was 9.5 years (ranged from 1.2 to 20 years, DP 5.5). Refraction was available in 26 patients, from which 88.5 percent (23 patients) were hyperopic, and 53.8 percent (14 patients) presented astigmatism. Best corrected visual acuity was available in 18 patients and the mean was 0.45 logMAR (DP 0.68). The mean spherical equivalent was +3.57 D (SD 2.46) and intraocular pressure was 17 mmHg (SD 3.9). The most common findings were: eyelid thickening in 24.1 percent (7 patients); corneal opacity in 55.2 percent of cases (16 patients); optic nerve atrophy in 23.1 percent (6 patients); and radial folds in the retina in 24 percent (7 patients). The fundus was examined in 26 out of 29 patients because corneal opacity avoided the exam in 3 of them. The average thickness of the complex sclera-retina-choroid (SRC was 1.78 mm (SD 0.51). CONCLUSION: The most prominent ophthalmologic findings were eyelid thickening, decreased visual acuity, high hyperopia, corneal opacity, perimacular radial folds in the retina and optic nerve atrophy.
Assuntos
Criança , Feminino , Humanos , Masculino , Opacidade da Córnea/etiologia , Mucopolissacaridoses/complicações , Atrofia Óptica/etiologia , Erros de Refração/etiologia , Opacidade da Córnea/diagnóstico , Pressão Intraocular/fisiologia , Atrofia Óptica/diagnóstico , Prevalência , Erros de Refração/diagnóstico , Índice de Gravidade de Doença , Acuidade Visual/fisiologiaRESUMO
Whey is a co-product of processes for the production of cheese and casein that retains most of the lactose content in milk. World production of whey is estimated around 200 million tons per year with an increase rate of about 2 percent/per year. Milk production is seasonal, so surplus whey is unavoidable. Traditionally, whey producers have considered it as a nuisance and strategies of whey handling have been mostly oriented to their more convenient disposal. This vision has been steadily evolving because of the upgrading potential of whey major components (lactose and whey proteins), but also because of more stringent regulations of waste disposal. Only the big cheese manufacturing companies are in the position of implementing technologies for their recovery and upgrading, so there is a major challenge in incorporating medium and small size producers to a platform of whey utilization, conciliating industrial interest with environmental protection within the framework of sustainable development. Within this context, among the many technological options for whey upgrading, transformation of whey components by enzyme biocatalysis appears as prominent. In fact, enzymes are green catalysts that can perform a myriad of transformation reactions under mild conditions and with strict specificity, so reducing production costs and environmental burden. This review pretends to highlight the impact of biocatalysis within a platform of whey upgrading. Technological options are shortly reviewed and then an in-depth and critical appraisal of enzyme technologies for whey upgrading is presented, with a special focus on newly developed enzymatic processes of organic synthesis, where the added value is high, being then a powerful driving force for industrial implementation.
Assuntos
Lactose , Leite/enzimologia , Oligossacarídeos/metabolismo , Prebióticos , beta-Galactosidase/metabolismo , Biocatálise , Esterificação , Enzimas/metabolismoRESUMO
Infantile GM1 gangliosidosis is caused by the absence or reduction of lysosomal beta-galactosidase activity. Studies conducted in Brazil have indicated that it is one of the most frequent lysosomal storage disorders in the southern part of the country. To assess the incidence of this disorder, 390 blood donors were tested for the presence of two common mutations (1622-1627insG and R59H) in the GLB1 gene. Another group, consisting of 26 GM1 patients, and the blood donors were tested for the presence of two polymorphisms (R521C and S532G), in an attempt to elucidate whether there is a founder effect. The frequencies of the R59H and 1622-1627insG mutations among the GM1 patients studied were 19.2% and 38.5%, respectively. The frequency of polymorphism S532G was 16.7%, whereas R521C was not found in the patients. The overall frequency of either R59H or 1622-1627insG was 57.7% of the disease-causing alleles. This epidemiological study suggested a carrier frequency of 1:58. Seven different haplotypes were found. The 1622-1627insG mutation was not found to be linked to any polymorphism, whereas linkage disequilibrium was found for haplotype 2 (R59H, S532G) (p < 0.001). These data confirm the high incidence of GM1 gangliosidosis and the high frequency of two common mutations in southern Brazil.
RESUMO
Introducción: Un campo de investigación creciente de la biología es la senescencia celular, mecanismo que ha sido asociado -bajo determinadas circunstancias- con la transformación maligna. Teniendo en cuenta la elevada incidencia de cáncer ovárico y su génesis preferencial a partir del epitelio superficial del ovario, así como la posibilidad de ocurrencia de una transición epitelio-mesenquimática, se evaluó, tanto el crecimiento in vitro de los fibroblastos del estroma cortical, como la actividad a pH 6 de la β-galactosidasa, enzima cuya expresión ha sido clásicamente considerada como marcador de senescencia replicativa. Metodología: 48 muestras de fibroblastos de la corteza ovárica provenientes de donantes sin antecedentes de cáncer fueron cultivadas en forma seriada hasta el final de su vida replicativa. Mediante el método quimioluminiscente, en cada pase fue cuantificada la actividad β-galactosidasa a pH 6. Como control se utilizaron cultivos de células del epitelio superficial ovárico de las mismas donantes. La actividad enzimática fue también evaluada en fibroblastos previamente inducidos a senescencia con peróxido de hidrógeno. Resultados: Las lecturas de actividad enzimática, analizadas en conjunto con la capacidad replicativa, indican que los cultivos de fibroblastos alcanzaron el estado senescente hacia los pases 4-5, lo que también ocurrió con las células epiteliales. Los fibroblastos inducidos a senescencia mostraron valores variables de actividad enzimática. Conclusiones: La semejanza entre los fibroblastos y las células epiteliales en cuanto al inicio de la senescencia podría estar relacionado con la transición epitelio-mesenquimática que ha sido descrita como factor de riesgo de cáncer derivado del epitelio superficial ovárico. Valores bajos de actividad β-galactosidasa podrían sugerir que, en algunos casos, ocurrió inactivación de las vías de respuesta al estrés oxidativo.
Introduction: A growing biological research field is the cellular senescence, a mechanism that has been associated, under certain circumstances, with malignant transformation. Given the high incidence of ovarian cancer and its main origin from the ovarian surface epithelium, as well as the possibility that an epithelial-mesenchymal transition occurs, we evaluated both the in vitro growth of stromal fibroblasts from the ovarian cortex and their β-galactosidase activity at pH 6, enzyme whose expression is considered as a marker of replicative senescence. Methods: 48 samples of ovarian cortical fibroblasts from donors without a history of cancer were serially cultured until the end of their replicative life. β-galactosidase activity at pH 6 was quantified in each passage by the chemiluminiscent method. As control, we used ovarian epithelial cell cultures from the same donors. The enzyme activity was also evaluated in fibroblasts previously induced to senescence by exposure to hydrogen peroxide. Results: The analysis of the enzyme activity and the replicative capacity taken together showed that the fibroblast cultures reached the senescent state at passages 4-5, as what happened with the control epithelial cells. Fibroblasts induced to senescence showed high variability in the values of enzymatic activity. Conclusions: The similarity between both types of cells in reaching the senescent state deserves to be taken into account in relation to the epithelial-mesenchymal transition that has been proposed to explain their behavior in the genesis of cancer arising from ovarian surface epithelium. Low β-galactosidase activity values at pH 6 would suggest possible inactivation of the response pathways to oxidative stress.
Introdução: um campo de pesquisa crescente da biologia é a senescência celular, mecanismo que tem sido associado, sob determinadas circunstancias, com a transformação maligna. Tendo em conta a elevada incidência de câncer ovariano e sua gênese preferencial a partir do epitélio superficial do ovário, assim como a possibilidade de ocorrência de uma transição epitélio-mesenquimática, se avaliou, tanto o crescimento in vitro dos fibroblastos do estroma cortical, como a atividade a pH 6 da β-galactosidase, enzima cuja expressão tem sido classicamente considerada como marcador de senescência replicativa. Metodologia: 48 amostras de fibroblastos do córtex ovariano provenientes de doadores sem antecedentes de câncer foram cultivadas em forma seriada até o final de sua vida replicativa. Mediante o método quimioluminescente, em cada passe foi quantificada a atividade β-galactosidase a pH 6. Como controle utilizou-se cultivos de células do epitélio superficial ovariano das mesmas doadoras. A atividade enzimática foi também avaliada em fibroblastos previamente induzidos a senescência com peróxido de hidrogeno. Resultados: as leituras da atividade enzimática, analisada em conjunto com a capacidade replicativa, indicam que os cultivos de fibroblastos alcançaram o estado senescente para os passes 4-5, o que também ocorreu com as células epiteliais. Os fibroblastos induzidos a senescência mostraram valores variáveis de atividade enzimática. Conclusões:as semelhanças entre os fibroblastos e as células epiteliais em quanto ao inicio da senescência poderia estar relacionado com a transição epitélio-mesenquimática que tem sido descrita como fator de risco de câncer derivado do epitélio superficial ovariano. Valores baixos de atividade β-galactosidase poderiam sugerir que, em alguns casos, ocorreu inativação das vias de resposta ao estresse oxidativo.
Assuntos
Humanos , Feminino , beta-Galactosidase , Neoplasias Ovarianas , Senescência Celular , Transição Epitelial-Mesenquimal , FibroblastosRESUMO
Infantile GM1 gangliosidosis is caused by the absence or reduction of lysosomal beta-galactosidase activity. Studies conducted in Brazil have indicated that it is one of the most frequent lysosomal storage disorders in the southern part of the country. To assess the incidence of this disorder, 390 blood donors were tested for the presence of two common mutations (1622-1627insG and R59H) in the GLB1 gene. Another group, consisting of 26 GM1 patients, and the blood donors were tested for the presence of two polymorphisms (R521C and S532G), in an attempt to elucidate whether there is a founder effect. The frequencies of the R59H and 1622-1627insG mutations among the GM1 patients studied were 19.2 percent and 38.5 percent, respectively. The frequency of polymorphism S532G was 16.7 percent, whereas R521C was not found in the patients. The overall frequency of either R59H or 1622-1627insG was 57.7 percent of the disease-causing alleles. This epidemiological study suggested a carrier frequency of 1:58. Seven different haplotypes were found. The 1622-1627insG mutation was not found to be linked to any polymorphism, whereas linkage disequilibrium was found for haplotype 2 (R59H, S532G) (p < 0.001). These data confirm the high incidence of GM1 gangliosidosis and the high frequency of two common mutations in southern Brazil.