Double mutation of Saccharomyces cerevisiae for enhanced β-D-fructofuranosidase fructohydrolase productivity and application of growth kinetics for parametric significance analysis

Abstract The kinetics of an extracellular β-D-fructofuranosidase fructohydrolase production by Saccharomyces cerevisiae in a chemically defined medium, i.e., sucrose peptone agar yeast extract at pH 6, was investigated. The wild-type was treated with a chemical mutagen, methyl methane sulfonate. Among the six mutants isolated, methyl methane sulfonate-V was found to be a better enzyme producing strain (52 ± 2.4a U/mL). The maximum production (74 ± 3.1a U/mL) was accomplished after at 48 h (68 ± 2.7a mg/mL protein). The mutants were stabilized at low levels of 5-fluoro-cytocine and the viable ones were further processed for optimization of cultural conditions and nutritional requirements. The sucrose concentration, incubation period and pH were optimized to be 30 g/L, 28 °C, and 6.5, respectively. The methyl methane sulfonate-V exhibited an improvement of over 10 folds in enzyme production when 5 g/L ammonium sulfate was used as a nitrogen source. Thin layer chromatography and high-performance liquid chromatography analysis illustrated the optimal enzyme activity supported by the higher rate of hydrolysis of sucrose into monosaccharides, particularly α-D-glucose and β-D-fructose. The values for Qp (2 ± 0.12c U/mL/h) and Yp/s (4 ± 1.24b U/g) of the mutant were considerably increased in comparison with other yeast strains (both isolates and viable mutants). The mutant could be exploited for enzyme production over a wider temperature range (26–34 °C), with significantly high enzyme activity (LSD 0.048, HS) at the optimal temperature.

Antitumoral activity of allicin from garlic / Actividad antitumoral de alicina de ajo

Epidemiological studies link increased garlic consumption with a reduced incidence of cancer in various human populations. Experimental carcinogenesis studies in animal models and in cell culture systems indicate that several allium-derived compounds exhibit inhibitory effects. To provide a better understanding of the effects of allium derivatives on the prevention of cancer, we examined allicin, the major component of garlic, for their effects on antitumoral activity in vitro and in L5178Y lymphoma bearing mice. We found that allicin decreased the growth of tumor cells whereas in vivo, the compound shown an antitumor effect in murine L5178Y lymphoma. Allicin enhanced the secretion of IL-2, IFN-gamma and TNF-alpha cytokines from mouse plasma. These cytokines are associated with the beneficial Th1 antitumor response, which is characteristic of effective cancer immunotherapies.

Los estudios epidemiológicos relacionan el aumento del consumo de ajo con una disminución en la incidencia de cáncer en diferentes poblaciones humanas. Los estudios experimentales de carcinogénesis en modelos animales y en los cultivos de células tumorales indican que varios compuestos derivados del ajo tienen efectos inhibitorios. A fin de proporcionar una mejor comprensión de los efectos de derivados del ajo en la prevención del cáncer, se evaluó la alicina el principal componente del ajo, en la actividad antitumoral in vitro y en ratones con linfoma. Se encontró que la alicina disminuyó el crecimiento de células tumorales, mientras que in vivo, el compuesto muestra un efecto antitumoral en el linfoma murino L5178Y. La alicina incrementó la secreción de las citocinas IL-2, interferón-gamma y TNF-alfa obtenidas de plasma de ratón con linfoma. Estas citocinas están asociadas con la respuesta antitumoral benéfica Th1, que es característica de inmunoterapias efectivas para el cáncer.

Ultrastructural study on the morphological changes to male worms of Schistosoma mansoni after in vitro exposure to allicin / Estudo ultraestrutural das alterações morfológicas de vermes machos de Schistosoma mansoni após exposição in vitro à alicina

INTRODUCTION: Garlic has a wide range of actions, including antibacterial, antiviral, antifungal, antiprotozoal and anthelmintic actions. This antiparasitic activity has been attributed to allicin, which is the main constituent of garlic. The present study aimed to investigate the in vitro activity of allicin on the tegument of adult Schistosoma mansoni worms using scanning electron microscopy. METHODS: Swiss Webster mice were infected with S. mansoni cercariae (100 per mouse) and sacrificed 50 days later to acquire the adult worms. These worms were collected by perfusion and placed in RPMI medium 1,640 at 37°C before transferring to RPMI media containing 0 (control), 5, 10, 15 and 20mg/mL of allicin, where they were incubated for 2h. The worms were fixed in 2.5 percent glutaraldehyde solution, washed twice, post-fixed in osmium tetroxide, washed twice and then dehydrated with ascending grades of ethanol. The samples were air-dried, mounted on stubs, gold coated in an ion sputtering unit and viewed using a scanning electron microscope. RESULTS: A concentration of 5mg/mL caused wrinkling in the tegument; a concentration of 10mg/mL resulted in changes to tubercles and loss or modification of spines. With 15 and 20mg/mL increasing damage to the tegument could be seen, such as vesicle formation and the presence of ulcers. CONCLUSIONS: These findings demonstrate the effect of allicin on adult S. mansoni worms and indicate that most of the changes occur at concentrations greater than that normally indicated for treatment.

INTRODUÇÃO: O alho apresenta uma ampla gama de ações, incluindo antibacteriana, antiviral, antifúngico, antiprotozoário e anti-helmíntico. Esta atividade antiparasitária tem sido atribuída à alicina, que é o principal constituinte do alho. O presente estudo teve como objetivo investigar a ação in vitro da alicina no tegumento de vermes adultos de Schistosoma mansoni utilizando a microscopia eletrônica de varredura. MÉTODOS: Camundongos Swiss Webster foram infectados com cercárias de S. mansoni (100 por camundongo) e sacrificados 50 dias depois para aquisição de vermes adultos. Estes vermes foram coletados por perfusão e colocados em meio RPMI 1.640 a 37°C antes de transferir para o meio RPMI contendo 0 (controle), 5, 10, 15 e 20mg/mL de alicina, onde eles foram incubados por 2h. Os vermes foram fixados em uma solução de glutaraldeído a 2,5 por cento, lavados duas vezes, pós-fixados em tetróxido de ósmio, lavados duas vezes e então desidratados em séries crescentes de etanol. As amostras foram secadas, montadas em stubs, metalizadas em ouro e visualizadas utilizando o microscópio eletrônico de varredura. RESULTADOS: A concentração de 5mg/mL causou o enrugamento do tegumento; a concentração de 10mg/mL resultou em alterações nos tubérculos e perda ou modificações nos espinhos. Com 15 e 20mg/mL crescentes danos no tegumento pode ser visto, tais como formação de vesículas e presença de úlceras. CONCLUSÕES: Esses resultados demonstram os efeitos da alicina nos vermes adultos de S. mansoni e indicam que a maioria das alterações ocorrem numa concentração maior do que a normalmente indicada para o tratamento.