1-Cyanocyclohexaneacetic acid (1-CHAA) is an important intermediate in the preparation of gabapentin. Regioselective biotransformation of 1-cyanocyclohexaneacetonitile (1-CHAN) into 1-CHAA, mediated by the immobilization of recombinant microbial nitrilase, is a novel and promising strategy for its production. In this study, a recombinant E. colt expressing Acidovorax facilis nitrilase was flocculated with polyethylenemine, followed by cross-linking with glutaraldehyde to obtain cross-linked cell aggregates (CLCAs). Under the optimum preparation conditions, the activity recovery of CLCAs reached 68%. Characterization of the CLCAs showed that the optimum pH was not affected by cross-linking, but cross-linking resulted in slightly different optimum temperature. The half-life of CLCAs at 45 degrees C was 266.5 h, which is 11.6-times higher than that of the free cells. Preparative-scale biotransformation of 1-CHAN to 1-CHAA at high substrate loading (up to 1.0 M) was performed using CLCAs as the biocatalyst in a 2L stirred reactor, resulting in an conversion of 91%, with a productivity of 244.5 g L-1 d(-1). The CLCAs also exhibited a high operational stability and even after 30 cycles of reaction, it retained > 80% of the initial conversion.