AbstractIn this study, β-1, 4-endoglucanase from Penicillium simplicissimum H-11 was purified to homogeneity using ammonium sulfate followed by Sephadex G-100 chromatography. The purity of the enzyme was confirmed by HPLC and 12% SDS-PAGE, indicating a single peak with a molecular mass of 33.2 kDa. This protein had mostly α-helix structures, as confirmed by FTIR spectrometry. The optimum pH and temperature were 3.2 and 60 °C with pH stability of 2.8~5.6 and temperature stability of 50 °C for 12 h and 4 h, respectively. A metal profile of the enzyme showed that Mg2+ and Sn2+ were strong activators, while Cu2+ was a strong inhibitor. An interesting feature of this enzyme is that it can effectively hydrolyze microcrystalline cellulose, filter paper, and CMC-Na, thus revealing both endo- and exo-glucanase features of the enzyme. The kinetic constants Km and Vmax were 14.881 mg/mL and 0.364 mg/mL/min, respectively, against CMC-Na as a substrate.