Response-surface methodology for the production and the purification of a new H2O2-tolerant alkaline protease from Bacillus invictae AH1 strain

This work deals with the optimization of the culture conditions of Bacillus invictae AH1 in order to increase the production level of the proteolytic activity. Response-surface methodology (RSM) was applied for the most significant fermentation parameters (concentration of wheat bran and K₂HPO₄/KH₂PO₄) that were earlier identified by Plackett–Burman Design from seven possible factors. A central composite design was used and the quadratic regression model of producing active protease was built. A maximum protease activity was reached and validated experimentally, using a maximum wheat bran concentration (50 g/L) with increased K₂HPO₄/KH₂PO₄ concentration (2.275 g/L). Protease production obtained experimentally coincident with the predicted value and the model was proven to be adequate. Interestingly, the use of RSM increased the protease production by four times (7,000 U/mL) using a low-cost substrate and a culture time of 40 hr, as compared to the standard culture conditions. In the second part of this study, a H₂O₂-tolerant alkaline protease produced from B. invictae AH1 with a molecular mass of about 41 kDa, noted P3, was purified by successive steps of ultrafiltration, gel filtration and ion exchange chromatography. The K _m and V_max values of the purified protease using casein, as substrate, were about 4 mg/mL and 27 μM/min, respectively. The highest enzyme activity was found at pH 9.0 and a temperature of 60°C. In addition, the enzyme showed a quasi-total stability against H₂O₂ (5% for 1 hr) and against most of the tested solid and liquid detergents, suggesting its eventual use in bio-detergent formulations.     

Effect of salinity on germination,phytase activity and phytate content in lettuce seedling

Seeds of four lettuce (Lactuca sativa L.) varieties (Romaine, Augusta, Vista and Verte) differing in their salt sensitivity were sown at 0 (Control), 50, 100 and 150 mM NaCl. The final germination percentage decreased with the increasing salinity and was annulated at the highest salt concentration in Vista and Verte, the most sensitive varieties. However, in the less sensitive ones, Romaine and Augusta, it was slightly modified at 50 and 100 mM NaCl and then decreased by 50% compared with the control, at 150 mM. The effects of NaCl 100 mM on seedling growth, phytase activities, phytate and inorganic phosphorus contents were studied in Romaine and Vista showing different behaviours towards salinity. Radicle and hypocotyl length and fresh and dry weights were reduced by salt treatment in both varieties. In addition, radicle phytase activity exhibited an increase in Romaine (less sensitive) and a decrease in Vista (more sensitive). In hypocotyl, this activity showed no difference with the control in the two varieties. However, in cotyledons, and during early hours after germination, salinity decreased phytase activity in both varieties whereas in the later hours (72–96 h) this activity reached the value of the control in Romaine. The enhancement of phytase activity was concomitant with an increase in orthophosphate content and a decrease in phytate reserve. These results suggest that salt presence in the medium delays Pi remobilization from phytate stock, but stimulates assimilation of phosphorus more than its accumulation in the organs of the two lettuce varieties.     

Design of novel granulopoietic proteins by topological rescaffolding

Computational protein design is rapidly becoming more powerful, and improving the accuracy of computational methods would greatly streamline protein engineering by eliminating the need for empirical optimization in the laboratory. In this work, we set out to design novel granulopoietic agents using a rescaffolding strategy with the goal of achieving simpler and more stable proteins. All of the 4 experimentally tested designs were folded, monomeric, and stable, while the 2 determined structures agreed with the design models within less than 2.5 Å. Despite the lack of significant topological or sequence similarity to their natural granulopoietic counterpart, 2 designs bound to the granulocyte colony-stimulating factor (G-CSF) receptor and exhibited potent, but delayed, in vitro proliferative activity in a G-CSF-dependent cell line. Interestingly, the designs also induced proliferation and differentiation of primary human hematopoietic stem cells into mature granulocytes, highlighting the utility of our approach to develop highly active therapeutic leads purely based on computational design.

De novo designed cytokines that activate the G-CSF receptor show that the receptor-binding information can be encoded onto stable, miniaturised protein scaffolds that possess potent granulopoietic activity; such novel proteins provide for ideal candidates for protein-based therapeutics.     

Seasonality of the endogenous activity rhythm in Talitrus saltator (Montagu) from a sandy beach in northeastern Tunisia

The endogenous activity of the sandhopper Talitrus saltator (Montagu) has been investigated in a population from a relatively exposed beach in northeast Tunisia. The animals were found to exhibit a nocturnal circadian rhythm of locomotor activity, with no evidence of a circatidal component. The rhythm was monitored in individual animals over the four seasons, under constant laboratory conditions and seasonal variation in free-running period; the signal-to-noise ratio and waveform of the rhythm have been investigated, as well as the incidence of rhythmic animals in the population. The variation in these rhythm parameters is considered in relation to changes in environmental conditions prevailing at the site of collection.