EFFICIENT THREE PHASE PARTITIONING OF MILK CLOTTING PROTEASE FROM Carica papaya LATEX AND ITS CHARACTERIZATION

Abstract

Papain is a cysteine protease derived from Carica papaya, which plays crucial role in various industries due to its proteolytic and milk clotting activity. The primary objective of the present research was to isolate, purify, and characterize the milk clotting protease (papain) found in the latex of the Carica papaya. The crude extract was put forward to three phase partitioning (TPP) with varied optimization parameters, including ammonium sulphate concentration (30-80%, w/v), crude extract to t-butanol ratio (1:0.5-1:2, v/v), and pH (4-9). Caseinolytic activity (CA) and protein content were used to determine the maximum activity recovery (%) and purification fold for both the intermediate phase and the aqueous phase. The effect of pH and temperature on activity and stability, as well as the effect of metal ions and inhibitors on the activity of isolated enzyme, was studied by assessing CA. The kinetic parameters of the purified enzyme were determined by measuring CA using different casein concentration ranging from 0 to 50 mg/ml. Furthermore, response surface methodology using central composite design was used to calculate the ideal milk temperature and pH for maximum milk clotting activity (MCA). The storage stability of the protease was assessed as well. The protease having the highest activity was found to be concentrated more at the aqueous phase (AP) with 70% ammonium sulphate precipitation, crude extract to t-butanol ratio of 1:0.75 (v/v), and pH 6 resulting 9.21 purification fold and 159.71% activity recovery. The enzyme activity was highest at 60°C and pH 6.0. It was discovered to be stable for 2 h between 40 to 70°C and 6.0 to 8.0 pH. The Vmax and Km values were found to be 1250.8 U/ml and 14.8 mg/ml respectively. The activity of purified protease was found to be enhanced by Ca2+ and Mg2+ metal ions and EDTA. A numerical optimization study demonstrated that the ideal temperature and pH of milk for maximum MCA were 55°C and pH 6.0, respectively. The observed optimum MCA was 809.19 U/ml. MCA of papain was found to be highly stable when stored under freezing (-20°C) as compared at 4°C for 3 weeks. This study showed that the TPP can be used to purify papain from Carica papaya latex economically, and it can also be employed as a vegetable coagulant for cheesemaking. Also, the optimum conditions for maximum enzyme activity enables industries to harness the full potential of enzymes in diverse applications, from manufacturing processes to bioengineering.