Three phase partitioning, a scalable method for the purification and recovery of cucumisin, a milk-clotting enzyme, from the juice of Cucumis melo var. reticulatus

Cucumisin [EC 3.4.21.25] was first purified from Cucumis melo var. reticulatus juice by three-phase partitioning (TPP). Optimum purification parameters of the TPP system were determined as 60% ammonium sulfate saturation with 1.0:1.25 ratio of crude extract: t-butanol at pH and temperature of 8.0 and 20 °C, respectively. Cucumisin was purified with 4.61 purification fold and 156% activity recovery. The molecular weight of the recovered cucumisin was determined as 68.4 kDa and its isoelectric point is 8.7. Optimum pH and temperature of cucumisin were pH 9.0 and 60-70 °C, respectively. The protease was very stable at 20-70 °C and a pH range of 2.0-12.0. Km and Vmax constants were 2.24 ± 0.22 mgmL−1 and 1048 ± 25 μ Mmin−1, respectively. The enzyme was stable against numerous metal ions and its activity was highly enhanced by Ca2+, Mg2+, and Mn+2. Cucumisin activity was 2.35-folds increased in the presence of 5 mM of CaCl2. It was inactivated by Co2+, Cd2+, Zn2+ and Fe2+ and dramatically by PMSF. Cucumisin milk-clotting activity was highly stable when stored under freezing (−20 °C) compared at 4 °C and 25 °C. Finally, TPP revealed to be a useful strategy to concentrate and purify cucumisin for its use as a milk-clotting enzyme for cheese-making.