EFFECT OF EXTRACTION METHODS ON PROTEASE YIELD AND PROTEOLYTIC ACTIVITIES OF PROTEASE ENZYME FROM DIFFERENT MATURITY STAGES OF STARFRUIT (Averrhoa carambola)

Abstract

The main objective of this work was to compare the effect of extraction methods (ammonium sulphate, acetone and acetone TCA precipitation) on protease yield from starfruit of different maturity stages (unripe, semiripe and ripe). Proteolytic activity (PA) and protein were measured spectrophotommetrically. Optimum hydrolysis condition: temperature and pH of protease for maximum proteolytic activity was determined by response surface methodology. Protease were incubated for 12 h in different buffers in the pH range of 3.5 to 8.5 before the determination of proteolytic activity. In order to determine the effect of temperature, the protease was incubated at different temperatures in the range of 40 to 90°C. Storage stability of the proteases were also determined for one week storage period at <4°C. Higher yield and proteolytic activity were observed for protease using ammonium sulfate (40%) than acetone and acetone with TCA precipitations. As for maturity stages, PA and protein concentration yield from unripe stage were significantly (p<0.05) higher than those of semi ripe and ripe. Numerical optimization study revealed that the optimum temperature and pH for proteolytic activity were 65°C and 6.5 respectively. The optimized proteolytic activity was found to be 0.862±0.05 units/ml. Enzyme activity was significantly decreased during 7 days storage period at <4°C. This study suggested that ammonium sulfate precipitation is effective method for extraction of starfruit protease compared to acetone and acetone with TCA precipitations. In order to obtain higher protease activity, unripe starfruit will be better raw material for protease production.