Absidia corymbifera AS2 has been previously screened for effective biotransformation of astragalosides since it is able to catalyze the hydrolysis of acetyl ester moieties. In this study, an acetyl esterase from A. corymbifera AS2 was purified and its catalytic pathways were investigated. The purified enzyme was monomeric, with a molecular mass of36 kDa, and with optimal activity observed at pH 8.0 and 35 degrees C. It was stable within pH 7.0-9.5 and at temperatures lower than 45 degrees C. The K-m and V-max values for p-nitrophenyl acetate was estimated to be 3.76 and 17.64 mmol (min mg)(-1), respectively. We found that this enzyme can hydrolyze the acetyl groups at positions O-2 or O-3 of xylopyranosyl residue at the C-3 position of AS-I, isoAS-I, AS-II and isoAS-II, and convert these all to ASI. The pathways of deacetylation catalyzed by this enzyme were also clarified for the first time: AS-II -> ASI, isoAS-II -> AS-II -> ASI, AS-I ->(AS-II, isoAS-II)-> ASI and isoAS-I -> AS-II -> ASI. In summary, an acetyl esterase from A. corymbifera AS2 was extracted, which showed unique enzymatic characteristics and enabled clarification of the biotransformation pathways of astragalosides. This enzyme has potential industrial applications, especially for utilizing abundant astragaloside precursors for the production of rare ASI. (C) 2014 Elsevier Ltd. All rights reserved.