Purification, cloning, sequencing and over-expression in Escherichia coli of a regioselective aliphatic nitrilase from Acidovorax facilis 72W

A regioselective aliphatic nitrilase from Acidovorax facilis 72W was purified and characterized, and the corresponding gene was cloned and sequenced. This nitrilase gene was over-expressed in Escherichia coli, generating a microorganism that efficiently and regioselectively catalyzes the conversion of aliphatic dinitriles to cyanocarboxylic acids. The high yields obtained, mild reaction conditions used, and robustness observed make this biocatalyst suitable for industrial applications.

[1]  C. Brenner,et al.  The nitrilase superfamily: classification, structure and function , 2001, Genome Biology.

[2]  M. Payne,et al.  Engineering Pichia pastoris for stereoselective nitrile hydrolysis by co-producing three heterologous proteins , 1999, Applied Microbiology and Biotechnology.

[3]  R. Fallon,et al.  Chemoenzymic Production of Lactams from Aliphatic α,ω-Dinitriles. , 1998 .

[4]  Y. Asano,et al.  Novel heme-containing lyase, phenylacetaldoxime dehydratase from Bacillus sp. strain OxB-1: purification, characterization, and molecular cloning of the gene. , 2000, Biochemistry.

[5]  R. Fallon,et al.  A Gram-negative bacterium producing a heat-stable nitrilase highly active on aliphatic dinitriles , 1999, Applied Microbiology and Biotechnology.

[6]  J. Crouzet,et al.  Aliphatic nitrilase from a soil-isolated Comamonas testosteroni sp.: gene cloning and overexpression, purification and primary structure. , 1995, Gene.

[7]  R. Fallon,et al.  Chemoenzymatic production of 1,5-dimethyl-2-piperidone , 2001 .

[8]  Lu Lu,et al.  The genetic structure of recombinant inbred mice: high-resolution consensus maps for complex trait analysis , 2001, Genome Biology.

[9]  H. Yamada,et al.  Nitrilase from Rhodococcus rhodochrous J1. Sequencing and overexpression of the gene and identification of an essential cysteine residue. , 1992, The Journal of biological chemistry.

[10]  M. Payne,et al.  Over-production of stereoselective nitrile hydratase from Pseudomonas putida 5B in Escherichia coli : activity requires a novel downstream protein , 1997, Applied Microbiology and Biotechnology.

[11]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[12]  D. Stalker,et al.  Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the bxn gene. , 1988, The Journal of biological chemistry.

[13]  R. Fallon,et al.  Chemoenzymic Production of Lactams from Aliphatic α,ω-Dinitriles , 1998 .

[14]  D. Cowan,et al.  Biochemistry and biotechnology of mesophilic and thermophilic nitrile metabolizing enzymes , 1998, Extremophiles.

[15]  H. Yamada,et al.  Primary structure of an aliphatic nitrile-degrading enzyme, aliphatic nitrilase, from Rhodococcus rhodochrous K22 and expression of its gene and identification of its active site residue. , 1992, Biochemistry.

[16]  M. Payne,et al.  Optimization of an immobilized-cell biocatalyst for production of 4-cyanopentanoic acid , 2002 .

[17]  H. Yamada,et al.  Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile: cloning of the Alcaligenes gene and site-directed mutagenesis of cysteine residues. , 1993, Proceedings of the National Academy of Sciences of the United States of America.