ORIGINAL RESEARCH PAPER Efficient expression in E. coli of an enantioselective nitrile hydratase from Rhodococcus erythropolis Liya Song Æ Hong-Jie Yuan Æ Lee Coffey Æ John Doran Æ Mei-Xiang Wang Æ Shijun Qian Æ Catherine O’Reilly Received: 26 June 2007 / Revised: 7 November 2007 / Accepted: 9 November 2007 / Published online: 28 November 2007 Ó Springer Science+Business Media B.V. 2007 Abstract The genes encoding an enantioselective nitrile hydratase (NHase) from Rhodococcus ery- thropolis AJ270 have been cloned and an active NHase has been produced in Escherichia coli. Maximal activity was found when the genes encoding the a- and b-subunits were transcribed as one unit and the gene encoding the P44k activator protein as a separate ORF on a single replicon. Addition of n-butyric acid and FeSO 4 could improve NHase activity. Coexpression of the GroEL-GroES chaper- one proteins increased activity in the absence of P44k protein but had no effect in the presence of P44k. The recombinant enzyme was highly enantio- selective in the synthesis of S-(+)-3-benzoyloxy- 4-cyanobutyramide from the prochiral substrate 3-benzoyloxyglutaronitrile. Keywords Activator protein Á Enantioselectivity Á GroES-GroEL Á Nitrile hydratase Á Rhodococcus erythropolis Introduction Nitrile hydratase (NHase, E C 4.2.1.84) is an enzyme catalyzing the hydration of nitriles to the correspond- ing amides. This enzyme has been the subject of considerable interest largely due to its potential as an industrial biocatalyst. Currently, NHase, produced by different microorganisms, is used in the synthesis of acrylamide, nicotinamide and 5-cyanovaleramide (Hann et al. 1999; Kobayashi and Shimizu 2000). Nitrile hydratase is a metalloenzyme containing non-heme iron (Fe-type NHase) or non-corrinoid cobalt (Co-type NHase). The enzyme consists of two subunits, a and b, each with a Mr of about 23 kDa. Nitrile hydratase genes have been cloned from variety of microorganisms. In most organisms the NHase genes are linked to a gene encoding an amidase which will convert the amide product of the NHase to the corresponding carboxylic acid. While the Co- and Fe-type NHases from different organisms are significantly homologous at the amino acid level there are substantial differences at the genetic level (Kobayashi et al. 1991, 1993; Komeda et al. 1996; L. Song Á S. Qian Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China L. Song Graduate School of the Chinese Academy of Sciences, Beijing 100039, China L. Song Á H.-J. Yuan Á L. Coffey Á J. Doran Á C. O’Reilly (&) Department of Chemical and Life Sciences, Waterford Institute of Technology, Cork Road, Waterford, Ireland e-mail: coreilly@wit.ie H.-J. Yuan Á M.-X. Wang Laboratory of Chemical Biology, Center for Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China 123 Biotechnol Lett (2008) 30:755–762 DOI 10.1007/s10529-007-9611-3