Introduction: It is widely accepted that ion-pair increases rigidity and thermostability. There are numerous studies on ion-pairs and thermostability, but none are available about the effect of ion-pair on the activity of enzymes. This paper studies whether an ion-pair allows flexible movement in an enzyme molecule and affects its activity. Materials and methods: Ion-pairs are designed at the alpha-helix region of a Bacillus circulans xylanase, and they are far from the active-sites (23.85-25.15 angstrom). Two ion-pairing mutations are situated at the C-terminus (D151/E151-K154 ion-pairs) of the helix. One mutation is double-site (F48R-N151D), which introduces both the tertiary (R48-D151) and intra-helical (D151-K154) ion-pairs. Results and discussion: All of the mutants enhanced the catalytic efficiency against xylan (1.66-3.58 times). The double-site mutation showed a synergistic effect on the activity. Overall, the ion-pairs decreased the flexibility (increased rigidity) of the alpha-helix region and increased the active-site flexibility. The ion-pairs were destabilizing and surface-located; this means that the weaker destabilizing ion-pair still allows flexible movement in the active-site. There is higher mobility of the strand B4 where the active site residue E172 is located. Moreover, the residues lining the active-site cleft (strand B8) showed increased flexibility upon substrate binding. Conclusion: Increase in the activity was due to the increase in active-site flexibility and increased mobility of the residues lining the active-site cleft (strand B8). (C) 2013 Elsevier Ltd. All rights reserved.