Fast activated charcoal prepurification of Fusarium solani ß-glucosidase for an efficient oleuropein bioconversion.

Fungal ß-glucosidases were extensively studied regarding their various potential biotechnology applications. Here, we report the selection of Fusarium solani strain producing high yield of ß-glucosidase activity. The effect of some factors on ß-glucosidase production was studied including: Initial pH, medium composition, concentration of carbon and nitrogen sources, and particle size of raw substrates. The optimal enzyme production was obtained with 4 units of pH. The highest ß-glucosidase activity was produced on 4% wheat bran (WB) as raw carbon sources, reaching 5 U/mL. A positive correlation between WB particle size and the ß-glucosidase production level was settled. The last one was enhanced to 13.60 U/mL in the presence of 0.5% (w/v) of ammonium sulfate. Interestingly, the activated charcoal was used as an inexpensive reagent enabling a rapid and efficient purification prior step that improved the enzyme-specific activity. Eventually, F. solani ß-glucosidase acts efficiently during the bioconversion process of oleuropein. Indeed, 82.5% of oleuropein was deglycosylated after 1 hr at 40°C. Altogether, our data showed that the ß-glucosidase of F. solani has a potential application to convert oleuropein to ameliorate food quality.

Selection of cell death-deficient p53 mutants in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae is a useful system for the detection and transcriptional evaluation of mutant p53 in cancer. In previous work we showed that the overexpression of wild-type p53 induces yeast cell death on minimal medium; however, the R248W p53 mutant was completely inactive, and we suggested that ROS production is a key event in p53-induced yeast cell death. In this study we explored the effect of other p53 mutants, such as the hot-spot mutant R282W and the double mutant N268S::I332V. Unexpectedly, both mutants behaved inversely to R248W, as they completely inhibited yeast growth on minimal medium and induced ROS production. This phenotype 'yeast cell death on minimal medium' allowed for the subsequent screening of intragenic p53-inactivating mutations. In all cases, the 'revertant yeast clones' display a complete p53 inactivation through either gross deletion or nonsense mutations. More interestingly, missense mutations were also found: the deletion of I255 or substitution of R337G completely inactivated the p53 mutant R282W in the yeast context. Taken together, these results suggest that p53 tumour-derived mutants could be classified according to their ability to induce yeast cell death and not uniquely by their transcriptional activity on a selected target reporter gene.