Acetyl esterases are important in the complete degradation of acetylated polysaccharides, such as pectins and xylans. We isolated the gene encoding acetyl xylan esterase (AfAXE) from a genomic lambda library of Aspergillus ficuum. We cloned the corresponding cDNA by RT-PCR. The Afaxe gene contained two introns, one TATAA box, and two CAAT-like boxes. The transcription initiation site was 61 bp upstream of the start codon. The deduced amino acid sequence consisted of a putative 28-amino acid leader peptide and a mature protein with an estimated molecular mass of 29.5 kDa. The nearest homolog of the cloned gene was acetyl xylan esterase of A. niger. The cloned gene was placed in a Pichia expression vector and expressed in Pichia pastoris. The culture filtrate of the transformant liberated acetyl moieties from p-nitrophenyl acetate and its activity reached 75.8 IU/ml, which was over 100-fold greater than the activity of the native enzyme expressed in A. ficuum. The cloned enzyme catalyzed the release of acetic acid from acetylated hardwood xylan, confirming that the cloned gene encoded an acetyl xylan esterase of A. ficuum. The native and recombinant acetyl xylan esterases were purified from the culture filtrates of A. ficuum and P. pastoris, respectively. Both enzymes had approximately the same optimal temperature (37degreesC) and pH (7.0). The recombinant protein had greater tolerance for alkaline conditions (pH greater than or equal to 7.0), but was less thermostable above 55degreesC.