THREE PHASE PARTITIONING OF MILK COAGULATING ENZYME FROM KIWIFRUIT (Actinidia deliciosa) AND ITS CHARACTERIZATION

Abstract

The main objective of this study was to purify and characterize milk clotting protease from kiwifruit (Actinidia deliciosa). The kiwifruit dialyzed crude extract (CE) was subjected to three phase partitioning (TPP) in varying purification parameters of ammonium sulfate concentration (30-80%, w/v), ratio of CE to tert-butanol (1.0:0.5-1.0:2.0, v/v) and pH (4.0 8.0). The activity recovery and purification fold for intermediate phase (IP) and aqueous phase (AP) were determined using protein content and caseinolytic activity (CA). The impact of pH, temperature, metal ions and inhibitors on the enzyme activity was studied. The kinetic parameters were evaluated using casein concentrations ranging from 0 to 30 mg/mL. Optimum milk temperature and pH for maximum milk clotting activity (MCA) were determined by using response surface methodology (RSM). The storage stability was assessed by measuring MCA. The optimal purification conditions for TPP were determined to be 40% ammonium sulfate concentration, 1.0:0.75 ratio of CE to tert-butanol, and 6.0 pH. The highest activity was found to be concentrated at IP resulting 3.6 purification fold and 122% recovery. The enzyme activity was significantly (p<0.05) highest at 50°C and pH 7.0. The Vmax and Km values were 121.9 U/min and 3.2 mg/ml, respectively and the enzyme followed Michaelis Menten kinetics. The enzyme activity was increased by Ca2+ ions and decreased by iodoacetamide (IAA). RSM optimization study revealed that the optimum milk temperature and pH for maximum MCA were 55°C and 6.25, respectively. The MCA of purified protease was highly stable when kept at frozen condition (-20°C). Hence, this study revealed that TPP can be used as an economical method for the purification of kiwifruit protease and can be used as an alternative source of rennet for cheesemaking from plant source.