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

The cloned 9.4-kb insert of plasmid pNHJ20L containing low-molecular-m ass nitrile hydratase (L-NHase) gene from Rhodococcus rhodochrous J1 [ Kobayashi, M. et al. (1991) Biochim. Biophys. Acta 1129, 23-331 was di gested with various restriction enzymes, and the trimmed fragments wer e inserted into pUC18 or pUC19. A 1.96-kb EcoRI-SphI region located 1. 9-kb downstream of the L-NHase gene was found to be essential for the expression of amidase activity in Escherichia coli; the gene arrangeme nt of the amidase and the NHase in R. rhodochrous J1 differed from tho se in Rhodococcus species including N-774 and Pseudomonas chlororaphis B23. The nucleotide-determined sequence indicated that the amidase co nsists of 515 amino acids (54626 Da) and the deduced amino acid sequen ce of the amidase had high similarity to those of amidases from Rhodoc occus species including N-774 and P. chlororaphis B23 and to indole-3- acetamide hydrolase from Pseudomonas savastanoi. The amidase gene modi fied in the nucleotide sequence upstream from its start codon expresse d 8% of the total soluble protein in E. coli under the control of lac promoter. The level of amidase activity in cell-free extracts of E. co li was 0.468 unit/mg using benzamide as a substrate. This amidase was purified to homogeneity from extracts of the E. coli transformant with 30.4% overall recovery. The molecular mass of the enzyme estimated by HPLC was about 110 kDa and the enzyme consists of two subunits identi cal in molecular mass (55 kDa). The enzyme acted upon aliphatic amides such as propionamide and also upon aromatic amides such as benzamide. The apparent K(m) values for propionamide and benzamide were 0.48 mM and 0.15 mM, respectively. This amidase was highly specific for the S- enantiomer of 2-phenylpropionamide, but could not recognize the config uration of 2-chloropropionamide. It also catalyzed the transfer of an acyl group from an amide to hydroxylamine to produce the corresponding hydroxamate.