An extracellular alpha-galactosidase (EC 3.2.1.22) from Penicillium sp. F63 CGMCC 1669, designated Agl1, was purified to homogeneity by means of fast protein liquid chromatography (FPLC) with a molecular mass of 82 kDa on SDS-PAGE while 330 kDa on native gradient PAGE. Based on the partial amino acid sequences of the purified protein, Agl1 gene encoding the alpha-galactosidase was cloned and sequenced. The ORF of Agl1 consisted of 2205 nucleotides encoding a protein of 735 amino acids including 21 residues of a putative signal peptide in its N-terminal. Agl1 was intron-less, and a GUG codon was considered the start codon of the gene. Agl1 possessed the highest sequence identity of 69.5% with alpha-galactosidase AglC from Aspergillus niger and showed a little homology to those of Penicillum purpurogenum and Penicillium simplicissimum reported previously, namely, 7.5% and 8.0%, respectively. The mature protein had been expressed extracellularly in Pichia pastoris with a yield of 111 U/ml in a 3-1 fermenter. The optimum pH and temperature of the recombinant a-galactosidase were 5.0 and 40 degrees C, respectively. The recombinant alpha-galactosidase hydrolyzed the release of galactose from natural oligosaccharides, such as melibiose, raffinose, and stachyose. To our knowledge, this is the first report of gene cloning for Penicillium alpha-galactosidase belonging to family 36 of glycosyl hydrolases and expression in Pichia pastoris. (c) 2006 Elsevier Inc. All rights reserved.