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

Abstract

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 2.18×10³ min⁻¹ M⁻¹, respectively. The optimum temperature and pH were 25°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.