Partial purification and characterization of pectinmethylesterase from ripening guava (Psidium guajava L.) fruits

Employing the techniques of (NH)SO fractionation, ion exchange chromatography on DEAE cellulose and gel filtration through Sephadex G-100, pectinmethylesterase (EC 3.1.1.11) was purified from guava (Psidium guajava L.) fruits var. Hisar Safeda harvested at turning stage of maturity to 129-fold with 28% recovery. Molecular weight as determined by gel filtration was found to be 51 kDa and the enzyme preparation exhibited the same molecular weight under native (Native-PAGE) and denaturating conditions (SDS-PAGE) indicating that the enzyme was a monomer. With pectin as the substrate, it exhibited the Michaelis Menten kinetics with K value of 3.1 g l. The enzyme was found to be stimulated by Ca and Na and inhibited competitively by d-galacturonic acid with K value of 1.97 mM. The enzyme was completely inactivated by iodine while with diethyl pyrocarbonate and N-acetylimidazole, the enzyme was inhibited up to the extent of 56 and 45%, respectively. However, DTNB had no inhibitory effect whatsoever precluding the participation of any -SH group in the active centre. It is tentatively proposed that the enzyme has tyrosine and histidine residues at its active centre