We previously identified a number of mutations in Escherichia coli AppA2 phytase for enhancing its thermostability. The objective of the present study was to determine if these mutations (K46E, K65E, G103S, D112N, D144N, S209G, V227A, and G344D) could be sequentially added to further improve the thermostability of AppA2. Compared with the wild-type enzyme, two variants (D144N/V227A and D144N/V227A/G344D) out of the eight resulting mutants showed 15% enhancement in thermostability (as measured by residual activity after being heated at 80 degrees C for 10 min) and 4 to 5 degrees C increases in the melting temperatures (T-m). Based on the structural predictions with a highly homologous AppA phytase, the substitution D144N introduces a side-chain-side-chain hydrogen bond, thereby stabilizing the loop region (Gln137-Asn144), and the V227A substitution might eliminate structural hindrance between Val222 and Val227 that face each other in the beta-hairpin structure. In addition, overall catalytic efficiency (k(cat)/K-m) of the two mutants was also improved (P < 0.05) compared to the wild type. However, no further improvement in thermostability was observed by adding other mutations to D144N/V227A/G344D, which might result from unfavorable electrostatic interactions or structural perturbation. In conclusion, our results underscore the potential as well as difficulty of predicting synergistic effects of multiple mutations on thermostability within phytase.