Cloning and expression of the nitrile hydratase and amidase genes from Bacillus sp BR449 into Escherichia coli

A moderate thermophile, Bacillus sp. BR449 was previously shown to exhibit a high level of nitrile hydratase (NHase) activity when growing on high lev els of acrylonitrile at 55 degrees C. In this report, we describe the cloni ng of a 6.1 kb Sail DNA fragment encoding the NHase gene cluster of BR449 i nto Escherichia coti. Nucleotide sequencing revealed six ORFs encoding (in order), two unidentified putative proteins, amidase, NHase beta- and alpha- subunits and a small putative protein of 101 amino acids designated P12K. S pacings and orientation of the coding regions as well as their gene express ion in E. coli suggest that the beta-subunit, alpha-subunit, and P12K genes are co-transcribed. Analysis of deduced amino acid sequences indicate that the amidase (348 aa, MW 38.6 kDa) belongs to the nitrilase-related aliphat ic amidase family. and that the NHase beta- (229 aa, MW 26.5 kDa) and alpha - (214 aa, MW 24.5 kDa) subunits comprise a cobalt-containing member of the NHase family, which includes Rhodococcus rhodochrous J1 and Pseudomonas pu tida 5B NHases. The amidase/NHase gene cluster differs both in arrangement and composition from those described for other NHase-producing strains. Whe n expressed in Escherichia coli DH5 alpha, the subcloned NHase genes produc ed significant levels of active NHase enzyme when cobalt ion was added eith er to the culture medium or cell extracts. Presence of the P12K gene and ad dition of amide compounds as inducers were not required for this expression . (C) 2000 Elsevier Science Inc. All rights reserved.