Characterization and functional cloning of an aromatic nitrilase from Pseudomonas putida CGMCC3830 with high conversion efficiency toward cyanopyridine

摘要: Abstract Nitrilases have long been considered as an attractive alternative to chemical catalyst in carboxylic acids biosynthesis due their green characteristics and the catalytic potential nitrile hydrolysis. A novel nitrilase from Pseudomonas putida CGMCC3830 was purified homogeneity. pI value estimated be 5.2 through two-dimensional electrophoresis. The amino acid sequence of NH2 terminus determined. Nitrilase gene cloned CODEHOP PCR, Degenerate PCR TAIL-PCR. open reading frame consisted 1113 bp encoding a protein 370 acids. predicted showed highest identity (61.6%) Rhodococcus rhodochrous J1. enzyme highly specific toward aromatic nitriles such 3-cyanopyridine, 4-cyanopyridine, 2-chloro-4-cyanopyridine. It classified nitrilase. activity could reach up 71.8 U/mg with 3-cyanopyridine substrate, which prominent level among identified cyanopyridine converting enzymes. kinetic parameters Km Vmax for were 27.9 mM 84.0 U/mg, respectively. These data would warrant it candidate creating effective nitrilases applications synthesis further engineering.