Physico-chemical characterization of a milk-clotting fraction extracted from turkey (Meleagris gallopavo) proventriculus

Valorization of avian by-products such as turkey (Meleagris gallopavo) proventriculus in the preparation of milk clotting enzymes constitutes an alternative to commercial rennet. The clotting enzyme of Meleagris gallopavo has been prepared by ammonium sulfate at 50% saturation followed by purification by cation-exchange fast protein liquid chromatography (FPLC), and determination of the clotting protease activity performed on milk incubated at 35 °C. The clotting fractions are concentrated and dialyzed against Tris/HCl pH 7.0 through a membrane with cutoff 8000 Da, and separated by anion-exchange FPLC. The coagulating fractions obtained by cation-exchange chromatography are also separated by reversed-phase high-performance liquid chromatography, which revealed only one fraction able to coagulate milk. Electrophoresis analysis revealed a band with apparent molecular mass of 36.5 kDa and seemed to correspond to pepsin-like enzyme according to primary sequence alignment analyses. Maximal clotting activity was obtained for optimal conditions of temperature of 55 °C and pH 5.4.