Maroua Guiza, Mouna Sahnoun, Faical Brini, Mansour Haddad and Walid Saibi*
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating CO2 into the biosphere. In this work, we report the extraction, the purification of the Alfalfa Rubisco. Indeed, the purification steps of the last one lead to the purified protein composed of two subunits (13 kDa and 55 kDa) as indicated in SDS-PAGE. The Rubisco proteolytic process via proteinase-K was optimized. The Box-Behnken Response Surface Methodology was applied to optimize three factors for increasing peptide production. So, the proteinase-K concentration (6, 13 and 20 mg/mL), the reaction incubation time (6 h, 8 h and 10 h) and the reaction temperature (30°C, 43°C and 56°C) were selected for the experimental design. A best condition of 20 mg/mL of proteinase-K, 10 h of time incubation, and 30°C of temperature treatment resulted in a 79.83%. This yield was 2.66 times higher than the one achieved before optimization. The resulting hydrolysate was injected in gel filtration column coupled to HPLC giving rise to four fractions (F1, F2, F3 and F4) eluted respectively after 12.54 min, 12.47 min, 12.44 min and 12.60 min. Also, the ROS scavenging system was monitored like the CAT, SOD and also the DPPH capacity. Eventually, those findings will give birth to the opportunity to look for plausible biotechnological investigations.
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