Purification and kinetic characterization of polygalacturonase - 3 from palmyrah palm (Borassus flabellifer L)

Abstract

Polygalacturonase-3 was isolated and purified to homogeneity from palmyrah palm (Borassus flabellifer L.) fruit using Con A-Sepharose affinity column. The purified enzyme migrated as a single band on native and SDS–polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme was estimated to be 66 kDa by size elution chromatography. Optimum polygalacturonase activity as a function of pH and temperature was determined using polygalacturonic acid as substrate. Optimum pH and temperature values ranged between the pH 4.0–5.0 and temperature 30–40 °C. At the optimum pH and temperature, the Km and Vmax values were determined by Lineweaver–Burk method. The value Km (0.33 mM) reveals that polygalacturonase has significant reactivity towards polygalacturonic acid. The enzyme showed varied responses towards divalent and monovalent metal ions. Ca2+ activated the polygalacturonase-3 enzyme protein. Both teepol and cetyltrimethylammonium bromide inhibited polygalacturonase-3 activity by 44 %, while 2-mercaptoethanol stimulated the enzyme marginally.