Article Dans Une Revue International Journal of Biological Macromolecules Année : 2017

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

Résumé

Cucumisin [EC 3.4.21.25] was first purified from Cucumis melo var. reticulatus juice by three-phase parti-tioning (TPP). Optimum purification parameters of the TPP system were determined as 60% ammoniumsulfate 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 molecularweight of the recovered cucumisin was determined as 68.4 kDa and its isoelectric point is 8.7. OptimumpH and temperature of cucumisin were pH 9.0 and 60–70◦C, respectively. The protease was very stable at20–70◦C and a pH range of 2.0–12.0. Km and Vmax constants were 2.24 ± 0.22 mgmL−1and 1048 ± 25 Mmin−1, respectively. The enzyme was stable against numerous metal ions and its activity was highlyenhanced by Ca2+, Mg2+, and Mn+2. Cucumisin activity was 2.35-folds increased in the presence of 5 mM ofCaCl2. It was inactivated by Co2+, Cd2+, Zn2+and Fe2+and dramatically by PMSF. Cucumisin milk-clottingactivity was highly stable when stored under freezing (−20◦C) compared at 4◦C and 25◦C. Finally, TPPrevealed to be a useful strategy to concentrate and purify cucumisin for its use as a milk-clotting enzymefor cheese-making.

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hal-01578265 , version 1 (28-08-2017)

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Mohammed Gagaoua, Ferhat Ziane, Sabrina Nait Rabah, Nawel Boucherba, Amel Ait Kaki El-Hadef El-Okki, et al.. 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. International Journal of Biological Macromolecules, 2017, 102, pp.515-525. ⟨10.1016/j.ijbiomac.2017.04.060⟩. ⟨hal-01578265⟩
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