Kinetic studies of bromelain purified from Palembang pineapple (Ananas comosus [L.] Merr) using gel filtration chromatography and its activity as antiplatelet aggregation

Kinetic study and antiplatelet activity of bromelain purified from Palembang pineapple (Ananas comosus [L] Merr) were carried out. Bromelain was isolated from pineapple's core and continued by two steps fractionation. First, fractionation was done using ammonium sulphate and continued by gel filtration chromatography using Sephadex G-50. The crude enzyme isolated has a specific activity of 53.580 Units/mg. Fractionation using ammonium sulphate (20-50 %) produced an optimum activity of bromelain fraction with specific activity of 230.970 Units/mg and the purity was 8 times greater. Further purification by Sphadex G-50 chromatography, increased the specific activity to 431.548 Units/mg and the purity was 28 times from the crude enzyme. The purified bromelain fraction has an optimum activity at pH 7.0 and temperature of 37°C. Kinetical studies were done by determining its activities on the various substrate concentrations. By plotting the curves of Lineweaver-Burk, the Michaelis-Menten Constant, Km of 0.777 % and maximum rate, Vmax of 3.969 U/min were obtained. The antiplatelet activity test showed that bromelain fractions from each purification step have the ability as an antiplatelet agent. The highest antiplatelet activity was found in the purest enzyme fraction with an aggregate percent value of 48.64 % and IC₅₀ of was 88.314 μL/mL.