Characterization of a nitrilase from Arthrobacter aurescens CYC705 for synthesis of iminodiacetic acid

A nitrilase gene cyc705 from Arthrobacter aurescens CYC705 for synthesis of iminodiacetic acid (IDA) was cloned. This gene contained a 930 bp ORF, which encoded a polypeptide of 310 amino acids. A recombinant Escherichia coli BL21(DE3)/pET28a-cyc705 was constructed to achieve the heterologous expression of cyc705. This recombinant nitrilase was purified to homogeneity with a molecular weight of 36.7 kDa on SDS-PAGE and mass spectrometry, and characterized to be an oligomer of 14 subunits by gel permeation chromatography. Using iminodiacetonitrile (IDAN) as the substrate, the V-max, K-m, k(cat) and k(cat)/K-m were 9.05 U mg(-1), 43.17 mM(-1), 94.1 min(-1) and 2.18 x 10(3) min(-1) M-1, respectively. The optimum temperature and pH were 25 degrees C and 5.8. The suitable substrates for the purified nitrilase were short-chain aliphatic dinitriles. High concentration of IDAN could be hydrolyzed to IDA in a shorter time.