Aspergillus niger phytase (PhyA) has been used as a feed supplement to improve the bioavailability of phytate phosphorus to swine and poultry. However, it is unable to maintain its stability due to high temperature during the feed pelleting process. In this study, we performed site-directed mutagenesis in the Aspergillus niger N25 phyA (m) gene at residue 44I and 252 T, and they were replaced by glutamic acid and arginine. Single-site mutants I44E-PhyA and T252R-PhyA, as well as double-site mutant I44E/T252R-PhyA, were constructed to improve the thermostability of PhyA through hydrogen bondings and ionic interactions. The three mutant enzymes all showed more than 20 % improvement in thermostability compared to the wild-type enzyme after being heated at 80 A degrees C for 10 min. Their melting temperatures (T (m)) were increased by 1, 1, and 1.2 A degrees C, respectively. The k (m) values of I44E-PhyA, T252R-PhyA, and I44E/T252R-PhyA for sodium phytate were 78, 44, and 79 % lower (P < 0.05) than that of the wild-type enzyme. Overall catalytic efficiency (k (cat)/k (m)) of I44E-PhyA, T252R-PhyA, and I44E/T252R-PhyA was improved by 310, 155, and 84 % (P < 0.05) than that of the wild type, respectively. The catalytic efficiency did not seem to be negatively affected by the improvement in thermostability.