A glycosyl hydrolase family 5 endo-beta-mannanase gene (man5F63) was cloned from Penicillium freii F63 and overexpressed in Pichia pastoris. man5F63 contained an open reading frame of 1260 bp that encoded a polypeptide of 419 amino acids including a putative 18-residue signal peptide. The recombinant enzyme (rMan5F63) was secreted into the culture supernatant to near electrophoretic homogeneity with a high yield (1.1 g I-1 in flask). Its apparent molecular weight was approximately 72.0 kDa, 29.0 kDa higher than the theoretical molecular mass. rMan5F63 was optimal at pH 4.5 and 60 degrees C and exhibited good stability over a broad pH range from acidic to alkaline (>85.0% activity at pH 4.0-9.0, > 70.0% activity at pH 10.0 and 43.7% even at pH 12.0). The activity of rMan5F63 was significantly enhanced in the presence of Co2+, Cu2+, Mn2+ and beta-mercaptoethanol and was strongly inhibited by He and SOS. The specific activity, Km and V,m, values were 47.5 U mg(-1), 7.8 mg ml(-1) and 70.4 mu mol min(-1) mg(-1), respectively, for locust bean gum, and 40.3 U mg(-1), 2.3 mg ml(-1) and 61.7 mu mol min(-1) mg(-1), respectively, for konjac flour. All these favorable enzymatic properties make it cost-effective to commercialization and valuable in various industries. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.