Two novel acetylesterases from Pantoea dispersa, with low amino acid sequence identity between them, were expressed in Escherichia coli with a carboxyl-His(6) tail given by the expression plasmid, purified, and characterized. The purified proteins, named Est-1 and Est-2, had a molecular mass of 33 kDa and 37 kDa, respectively. Both proteins presented a modeled structure of homodimers with monomers presenting the alpha/beta -hydrolase fold, with the catalytic triad Ser-Asp-His present in the active site. The K-M for p-nitrophenyl acetate and V-max values found for Est-1 were of 1.4 +/- 0.2 mM and 8.66 +/- 0.59 mu mol/min and for Est-2 were of 0.36 +/- 0.077 mM and 6.13 +/- 0.56 mu mol/min, respectively. Both enzymes presented an optimum pH of 7.0. The optimum temperature for Est-1 was 40 degrees C and for Est-2 was 50 degrees C. The temperatures in which the enzymes Est-1 and Est-2 lost half of their activity (T-50) were 44.1 and 58.9 degrees C, respectively. SDS, EDTA, and PMSF significantly inhibited the enzymes. The two purified enzymes also presented activity against triacetin and were able to deacetylate the carbohydrates pectin and xylan, with higher activity against pectin. Thus, they could be considered as carbohydrate esterases.