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1-Cyanocyclohexaneacetic acid is the key intermediate of gabapentin. A novel bioprocess catalyzed by amidase was developed for efficient production of 1-cyanocyclohexaneacetic acid from 1-cyanocyclohexaneacetamide, which can be prepared with high efficiency by nitrile hydratase-catalyzed regioselective hydration of 1-cyanocyclohexaneacetonitrile. Kinetic analysis and molecular docking of three recombinant amidase demonstrated that amidase (Pa-Ami) from Pantoea sp. was the most robust biocatalyst for hydrolysis of 1-cyanocyclohexaneacetamide with the kcat/Km value of 208.2 ± 16.2 mM−1 s−1. Some key parameters of the bioprocess, such as substrate loading, catalyst loading and product inhibition, were investigated. Enzymatic hydrolysis of 80 g/L of 1-cyanocyclohexaneacetamide was completed within 20 min using 1 g/L wet whole cells of recombinant Escherichia coli BL21, leading to high ratio of substrate to catalyst (S/C-ratio, 80) and high space-time yield (5794.7 gproduct L−1 d−1). These encouraging results indicated the great potential of Pa-Ami in practical production of gabapentin.