Inclusion of partly purified protease from Abrus precatorius Linn in Ca-alginate gel beads.

Abrus precatorius is an edible endemic plant in Cameroon. In the northern regions of Cameroon, the leaves of this plant are used as traditional medications, for the liquefaction of wort obtained after mashing of sorghum or millets for processing the local fermented beer called bili-bili, and to sweeten gruels made from red millet. Abrus precatorius has also being explored as a potential source of proteolytic enzymes. In this work, a partially purified protease from the leaves and stems of the plant was immobilized in a calcium alginate gel beads and the protease activity (PA), investigated using a central composite design plan under the conditions: alginate content (1-5%) (w/v), enzyme/alginate ratio (10-30%) (v/v), and CaCl2 concentration (100-400 Mmol/L). Results showed that the optimum activity was obtained for an alginate content of 1% (p/v), an enzyme/alginate ratio of 10% (v/v) and a CaCl2 concentration of 400 mmol/L. At the optimum, kinetic parameters of the immobilized enzyme (KM: 6.83 mg mL-1; Vmax: 46.95 g.L-1. min-1) were comparable to those of free enzyme (KM: 1.26 mg mL-1; Vmax: 153.85 g.L-1. min-1). The beads have been preserved at room temperature and reused for four days before loosing 88% of its activity. This indicates that the protease activity of the leaves and stems of A. precatorius can be immobilized in calcium alginate beads and preserve four days at ambient temperature.

Extraction, purification, and biochemical characterization of serine protease from leaves of Abrus precatorius.

A protease from fresh leaves of Abrus precatorius was purified using two classical chromatography techniques: ion-exchange (DEAE-Sepharose) and Gel filtration (Sephadex G-75). The purified protease showed a molecular weight of ∼ 28 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the purified protease was 8 and 40°C, respectively. The purified protease was stable throughout a wide temperature range from 10 to 80°C and pH from 2 to 12. Protease activity was inhibited in the presence of Co2+, Ni2+, Hg2+, and Zn2+ while its activity has increased in the presence of Ca2+ and Mg2+. The protease was highly specific to casein when compared to its specificity for gelatin, bovine serum albumin, hemoglobin, and defatted flour of Ricinodendron heudelotii. Its Vmax and Km determined using casein as a substrate were 94.34 U/mL and 349.07 µg/mL respectively. Inhibition studies showed that this purified protease was inhibited by both phenylmethane sulfonyl fluoride and aprotinin which are recognized as competitive inhibitors of serine proteases.