Nitrilases can hydrolyze nitriles to corresponding carboxylic acids in one single step, which have great potential as valuable biocatalysts for chemical synthesis. However, the poor thermostability of the nitrilases restrict their applications in industry. In this work, error-prone PCR and site-directed mutagenesis were utilized to improve the thermostability of nitrilases. Several mutants (AcN-Q339K, AcN-Q343K, AcN-T201F, AcN-T201W, AcN-T201L, AcN-T201I) were obtained with dramatically improved thermostability. The best mutant AcN-T201F/Q339K/Q343 Kexhibited about 14-fold longer half-life at 45 degrees C. The result of homology modeling suggested that the site 201, which was located on the "A" surface (the dimer interaction), played an important role in the oligomerization of nitrilase and the stabilization of substrate binding pocket. The Phe substitution on site 201 was selected in protein engineering of nitrilase LNIT5, which also demonstrated an improvement of thermostability. In addition, lysine substitution on Q339 and Q343 which brought positive charges to the a helix in the C-terminal region stabilized the surface.