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 68.4kDa 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±0.22mgmL-1 and 1048±25μ 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 5mM 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.