30 research outputs found

    Simple and efficient expression of Agaricus meleagris pyranose dehydrogenase in Pichia pastoris

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    Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH. Here, we evaluate recombinant protein production with expression hosts more suitable for genetic engineering. Expression in Escherichia coli resulted in no soluble or active PDH. Heterologous expression in the methylotrophic yeast Pichia pastoris was investigated using two different signal sequences as well as a codon-optimized sequence. A 96-well plate activity screening for transformants of all constructs was established and the best expressing clone was used for large-scale production in 50-L scale, which gave a volumetric yield of 223 mg L−1 PDH or 1,330 U L−1 d−1 in space–time yield. Purification yielded 13.4 g of pure enzyme representing 95.8% of the initial activity. The hyperglycosylated recombinant enzyme had a 20% lower specific activity than the native enzyme; however, the kinetic properties were essentially identical. This study demonstrates the successful expression of PDH in the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, the feasibility of large-scale production of the enzyme with this expression system together with a simplified purification scheme for easy high-yield purification is shown

    Impact of resolution on large-eddy simulation of midlatitude summertime convection

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    We analyze life cycles of summer time moist convection of a Large Eddy Simulation (LES) in a limited area setup over Germany. The goal is to assess the ability of the model to represent convective organization in space and time in comparison to radar data, and its sensitivity to daily mean surface air temperature. A continuous period of 36 days in May and June 2016, showing a considerable amount of convective rainfall which covers large parts of the domain on most of the days, is simulated with a grid spacing of 625 m. Using convective organization indices, and a tracking algorithm for convective precipitation events, we find that an LES with 625 m grid spacing tends to underestimate the degree of convective organization, and shows a weaker sensitivity of heavy convective rainfall to temperature as suggested by the radar data. An analysis of three days within this period that are simulated with finer grid spacing of 312 m and 156 m showed, that a grid spacing at the 100 m scale has the potential to improve the simulated diurnal cycles of convection, the mean time evolution of single convective events, and the degree of convective organization

    Relação entre excreção de creatinina e dieta, atividade física e volume urinårio, em crianças de 5 -| 12 anos de idade Relationship between creatinine excretion, diet, physical activity and urinary volume in children 5 to 12 years of age

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    Foi feito um estudo da excreção da creatinina e sua relação com a dieta, atividade fĂ­sica e volume urinĂĄrio em indivĂ­duos entre 5 -| 12 anos de idade, mantendo suas condiçÔes habituais de vida. A anĂĄlise qualitativa da dieta mostrou que a possibilidade de ingestĂŁo de creatina e creatinina ocorreu na hora do almoço. No que se refere Ă  atividade fĂ­sica, o perĂ­odo da manhĂŁ e o noturno foram de relativo repouso, concentrando-se as atividades fĂ­sicas no perĂ­odo da tarde. NĂŁo houve diferença, estatisticamente, significante entre a excreção mĂ©dia de creatinina dos perĂ­odos da manhĂŁ e da tarde e tambĂ©m entre a excreção mĂ©dia dos perĂ­odos diurno e noturno, sugerindo, conseqĂŒentemente, uma nĂŁo relação entre dieta, atividade fĂ­sica e excreção de creatinina. Houve uma diferença estatisticamente significante entre o volume mĂ©dio urinĂĄrio do perĂ­odo diurno e o do perĂ­odo noturno, sugerindo com isso uma independĂȘncia entre ele e a creatinina excretada. Encontrou-se, entretanto, uma correlação significante entre ambos que, possivelmente esteja ligada ao fato das amostras terem sido colhidas em Ă©pocas de frio intenso, em que os indivĂ­duos ingerem pouco lĂ­quido, e conseqĂŒentemente a urina estivesse com uma concentração elevada de seus diferentes constituintes.<br>A study on the relationship between creatinine excretion and diet, physical activity and urinary volume was carried out on children from 5 to 12 years of age, observing usual life conditions. Qualitative analysis of diet revealed that possibly ingestion of creatine and creatinine ocurred with the noon meal. Regarding physical activity the data showed that the peak of activity occurred in the afternoon. The differences between average excretion of creatine corresponding to morning and afternoon periods were not statistically significant, the same being observed when diurnal and nocturnal averages of excretion were considered. On the other hand, the average volumes corresponding to diurnal and nocturnal periods revealed a significant difference, which suggests independence between creatinine excretion and urinary volume. However data show a positive significant correlation between creatinine excretion and urinary volume when individual results were considered
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