R-Stereoselective amidase, a key enzyme responsible for the formation of chiral center of cilastatin, has been cloned from Delftia tsuruhatensis and expressed in Escherichia coli under T7 promoter. This recombinant amidase exhibits strict R-selectivity towards 2,2-dimethylcyclopropanecarboxamide (DMCPCA). The amidase activity of the engineered E. coli strain reached 2963 U/L in a 5-L bioreactor, which was 8.27 times higher than that of D. tsuruhatensis, and was further increased to 5255 U/L in a 100-L bioreactor. Using cell-free extract prepared from 1 kg (wet cell weight) of recombinant E. coli cells as catalyst, 60 kg of R,S-DMCPCA was resolved into S-DMCPCA (28.6 kg) and R-2,2-dimethylcyclopropanecarboxylic acid (R-DMCPCS 31.7 kg) in 18 h, and the enantiomeric excess (ee) value of S-DMCPCA reached 99.32%. During the purification process, 24.6 kg of S-DMCPCA was eluted from adsorption resin HZ801 with 80% (v/v) acetone, and then 20.5 kg of pure S-DMCPCA was obtained after concentration and crystallization, corresponding to a total yield of 34.2% from R,S-DMCPCA. Therefore, the industrial production process of S-DMCPCA was successfully established using recombinant R-amidase from D. tsuruhatensis. (C) 2010 Elsevier Ltd. All rights reserved.