Phytase is widely used as a feed additive in industry. It is important to investigate the thermal stability, optimal pH and temperature and protease resistance of phytases in application. We introduced random mutations in a protease-resistance phytase gene of Penicillium sp. using Mn2+-dITP random mutation method, and identified two mutants 2-28 (T11A, G56E, L65F, Q144H and L151S) and 2-249 (T11A, H37Y, G56E, L65F, Q144H, L151S and N354D) with improved thermal stability and optimal temperature and pH. The mutants retained their high resistance to pepsin. The catalytic activity at 37 C was up to 133.3 U and 136.6 U per mg protein with broad optimal temperature ranges of 37-55 degrees C and 37-50 degrees C, respectively. After a heat treatment at 100 degrees C for 5 min, the two mutant proteins retained about 72.81% and 92.43% of the initial activity, respectively. In addition, the optimal pH of mutant 2-249 was reduced to 4.8. All these improved properties made them more suitable to be used as feed additive in the feed industry than the present commercial phytases. Structure analysis suggested that the replacements of G56E, L65F, Q144H, and L151S improved the thermal stability of the protein by increasing new hydrogen bonds among the adjacent secondary structures. Moreover, the mutation of L151S enhanced the activity in the range of 37-70 degrees C and pH 2.5-7.0 by facilitating the interaction between the substrate and the catalytic centre. The substitution of N354D influenced the pH profile by weakening the bondage with the side chain of D353, which caused a pKa shift of the catalytic centre. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.