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

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Abstract
Pectin methylesterase A (EC 3.1.1.11), one of the pathogenicity factors of Erwinia chrysanthemi strain 3937, was purified to homogeneity using one-step chromatography on cross-linked pectate. The purified protein showed maximum activity at pH 8-9, 50°C, 50-100 mM monovalent cations or 5-10 mM 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 observed. A Km value of 0.03% and 0.051% was determined at pH 6 and 7.6, respectively, 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 during plant-pathogen interactions.Key words: soft rot disease, pectinase, affinity chromatography, polyclonal antibody.