Overproduction in Escherichia coli of the pectin methylesterase A from Erwinia chrysanthemi 3937: One-step purification, biochemical characterization, and production of polyclonal antibodies

Pectin methylesterase A (EC 3.1.1.21), one of the pathogenicity factors of Erwinia chrysanthemi strain 3937, was purified to homogeneity using one-ste p chromatography on cross-linked pectate. The purified protein showed maxim um activity at pH 8-9, 50 degrees C, 50-100 mM monovalent cations or 5-10 m M divalent cations, and on a 50% esterified pectin. A particular effect of Ca2+ and Zn2+ on PMEA activity, due to the formation of a pectin gel, was o bserved. A K-m value of 0.03% and 0.051% was determined at pH 6 and 7.6, re spectively, using the same substrate. Polyclonal antibodies raised against the PMEA from E. chrysanthemi strain 3937 were produced. It recognized PMEs from Erwinia species, but did not cross-react with PME of fungal or plant origin, and will therefore be a useful tool to immunolocalize the protein d uring plant-pathogen interactions.