Purification of ENOD8 proteins from Medicago sativa root nodules and their characterization as esterases

ENOD8 proteins were purified from alfalfa (Medicago sativa) root nodules. After extraction of ENOD8 proteins into an aqueous buffer, they were purified by ammonium sulfate precipitation, concanavalin A Sepharose chromatography, and ion-exchange chromatography. Purification was assessed by comparing silver stained SDS-PAGE gels to Western blots developed with a highly specific ENOD8 antibody. Multiple ENOD8 proteins that co-purified were found. ENOD8 proteins were found to have esterase activity, active on acetyl and butyrl esters but not longer chain aliphatic esters. Thus, ENOD8 proteins are unlikely to be lipases. Kinetic analysis showed that ENOD8 protein esterase activity exhibited Michaelis–Menten kinetics. Considering ENOD8 protein sequence similarity to an exopolygalacturonase/EP4/iEP4 and lanatoside 15′-O-acetylesterase with the results presented here predicts that ENOD8 substrates could be acetylated oligo- or polysaccharides.