Self-assembling amphipathic peptides (SAPs) are the peptides that can spontaneously assemble into ordered nano-structures. It has been reported that the attachment of SAPs to the N- or C-terminus of an enzyme can benefit the thermo-stability of the enzyme. Here, we discovered that the thermo-stability and product tolerance of nitrile hydratase (NHase) were enhanced by fusing with two of the SAPs (EAK16 and ELK16). When the ELK16 was fused to the N-terminus of beta-subunit, the resultant NHase (SAP-NHase-2) became an active inclusion body; EAKi6 fused NHase in the N-terminus of beta-subunit (SAP-NHase-1) and ELK16 fused NHase in the C-terminus of beta-subunit (SAP-NHase-10) did not affect NHase solubility. Compared with the deactivation of the wild-type NHase after 30 min incubation at 50 degrees C, SAP-NHase-1, SAP-NHase-2 and SAP-NHase-10 retained 45%, 30% and 50% activity; after treatment in the buffer containing 10% acrylamide, the wild-type retained 30% activity, while SAP-NHase-1, SAP-NHase-2 and SAP-NHase-10 retained 52%, 42% and 55% activity. These SAP-NHases with enhanced thermo-stability and product tolerance would be helpful for further industrial applications of the NHase. (c) 2014, The Society for Biotechnology, Japan. All rights reserved.