The beta-mannanase gene, man5C1, was cloned from Penicillium pinophilum Cl, a strain isolated from the acidic wastewater of a tin mine in Yunnan, China, and expressed in Pichia pastoris. The sequence analysis displayed the gene consists of a 1221-bp open reading frame encoding a protein of 406 amino acids (Man5C1). The deduced amino acid sequence of Man5C1 showed the highest homology of 57.8% (identity) with a characterized beta-mannanase from Aspergillus aculeatus belonging to glycoside hydrolase family 5. The purified rMan5C1 had a high specific activity of 1035 U mg(-1) towards locust bean gum (LBG) and showed highest activity at pH 4.0 and 70 C. rMan5C1 was adaptable to a wide range of acidity, retaining >60% of its maximum activity at pH 3.0-7.0. The enzyme was stable over a broad pH range (3.0 to 10.0) and exhibited good thermostability at 50 degrees C. The K-m and V-max a values were 5.6 and 4.8 mg mL(-1), and 2785 and 1608 mu mol min(-1) mg(-1), respectively, when LBG and konjac flour were used as substrates. The enzyme had strong resistance to most metal ions and proteases (pepsin and trypsin), and released 8.96 mg g(-1) reducing sugars from LBG in the simulated gastric fluid. All these favorable properties make rMan5C1 a promising candidate for use in animal feed. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.