Extraction, purification and characterization of a thermally stable aspartic protease from freshwater shrimp Gammarus sp. with a high catalytic efficiency

Abstract

In the curent study, a new aspartic protease is purified by pepstatin A affinity chromatography from freshwater amphipod Gammarus sp. The molecular weight of the enzyme was determined by SDS-PAGE which estimated to be 55 kDa. Optimal temperature and pH were 55 °C and 6, respectively. According to our results, calculated KM and Vmax for azocasein substrate were 0.05 μg mL−1 and 4.2 μmol min−1 while those of casein substrate were 0.006 μg mL−1 and 1.1 μmol min−1 respectively. The values of kcat constants for azocasein and casein substrates were calculated to be 2583 s−1 and 686 s−1 respectively. Moreover, the catalytic efficiencies (kcat/KM) for azocasein and casein substrates were 51,167 and 114,333 s−1 μg−1.ml respectively. The estimated ΔHD# , ΔGD# and ΔSD# were 4.221 kcal mol−1, 24.18 kcal mol−1 and 70 cal mol−1K−1respectively. The values of ΔGE−S# in the presence of azocasein and casein were −1.74 and 2.97 kcal mol−1 and those of ΔGE−T# for azocasein and casein were - 6.29 and - 6.76 kcal.mol−1respectively. Altogether, according to the obtained results, the new aspartic protease is thermally stable and catalytically efficient having a high affinity to milk casein as substrate and thus it has potential to be used in dairy industries for milk clotting.