Inhibitor effect of PCMB and PMSF towards partially purified product from pineapple core (Ananas comosus [L.] Merr) using gel filtration column chromatography method

Abstract

The research aims to isolate, purify, and examine the inhibitors effect of a partially purified product from pineapple core with Palembang type pineapple (Ananas comosus [L.] Merr). The isolation process obtained crude enzymes with specific activity 42.60 Units/mg. Purification of crude enzymes using fractionation method with ammonium sulfate obtained bromelain fraction with the highest specific activity at 0-50 % saturation level of ammonium sulfate, which is 230.22 Units/mg with a purity level of 5.40 times from its crude extract. Further purification of 0-50 % ammonium sulfate fraction by gel filtration chromatography method using DEAE-Sephadex G-50 matrix obtained enzyme with specific activity 275.46 Units/mg with purity level 6.50 times from its crude extract. The optimum substrate concentration was at a concentration of 1.0 % (w/v). The purified bromelain has a Michaelis-Menten (Km) constant value and the maximum speed of the enzymatic reaction (Vmax) i.e. 0.52 % (w/v) and 0.51 Units/min. The proteolytic activity of bromelain is strongly inhibited by PCMB compound and activated by PMSF at 0.1 mM concentration of 67.27 %.