Amidase coupled with low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1. Sequencing and expression of the gene and purification and characterization of the gene product.

摘要: The cloned 9.4-kb insert of plasmid pNHJ20L containing low-molecular-mass nitrile hydratase (L-NHase) gene from Rhodococcus rhodochrous J1 [Kobayashi, M. et al. (1991) Biochim. Biophys. Acta 1129, 23–33] was digested with various restriction enzymes, and the trimmed fragments were inserted into pUC18 or pUC19. A 1.96-kb EcoRI–SphI region located 1.9-kb downstream L-NHase found to be essential for expression amidase activity in Escherichia coli; arrangement NHase R. differed those species including N-774 Pseudomonas chlororaphis B23. nucleotide-deter-mined sequence indicated that consists 515 amino acids (54626 Da) deduced acid had high similarity amidases P. B23 indole-3-acetamide hydrolase savastanoi. The modified nucleotide upstream its start codon expressed 8% total soluble protein E. coli under control lac promoter. level cell-free extracts 0.468 unit/mg using benzamide as a substrate. This purified homogeneity transformant 30.4% overall recovery. molecular mass enzyme estimated by HPLC about 110 kDa two subunits identical (55 kDa). acted upon aliphatic amides such propionamide also aromatic benzamide. apparent Km values 0.48 mM 0.15 mM, respectively. highly specific S-enantiomer 2-phenylpropionamide, but could not recognize configuration 2-chloropropionamide. It catalyzed transfer an acyl group amide hydroxylamine produce corresponding hydroxamate.