Isolation and expression of a gene for a nitrilase from Acidovorax facilis 72W

An isolated nucleic acid fragment encoding a nitrilase enzyme, or a fragment thereof, selected from the group consisting of:

• (a) an isolated nucleic acid fragment selected from the group consisting of SEQ ID NO:
• 1, SEQ ID NO:
• 2, SEQ ID NO:
• 3, SEQ ID NO:
• 4, SEQ ID NO:
• 6, SEQ ID NO:
• 7, SEQ ID NO:
• 8, SEQ ID NO:
• 9, SEQ ID NO:
• 10, SEQ ID NO:
• 11, SEQ ID NO:
• 12, SEQ ID NO:
• 13, SEQ ID NO:
• 15 and SEQ ID NO:
• 16;
• (b) an isolated nucleic acid molecule that hybridizes with the isolated nucleic acid fragment of (a) under hybridization conditions of 6×SSC (1M NaCl), 40 to 45% formamide, 1% SDS at 37° C., and a wash in 0.5×to 1×SSC at 55 to 60° C.;
• and
• (c) an isolated nucleic acid fragment that is completely complementary to (a) or (b).

An isolated nucleic acid sequence encoding a nitrilase enzyme selected from the group consisting of SEQ ID NO:

• 4, SEQ ID NO:
• 13, SEQ ID NO:
• 15, and SEQ ID NO:
• 16.

A transformed microorganism selected from the group consisting of:

• (a) E. coli SW91 having the designation ATCC PTA-1175;
• (b) E. coli DH5α:
• pnit4 having the designation ATCC PTA-1176;
• (c) E. coli SS1001 having the designation ATCC PTA-1177;
• and
• (d) E. coli SS1002 containing plasmid pnitex2;
• and
• (e) E. coli SS1011 containing plasmid pnitex2.

A method of obtaining a nucleic acid fragment encoding all or a substantial portion of a nitrilase enzyme, the method comprising:

• (a) probing a genomic library with all or a portion of a nucleic acid fragment selected from the group consisting of SEQ ID NO:
• 1, SEQ ID NO:
• 2, SEQ ID NO:
• 3, SEQ ID NO:
• 4, SEQ ID NO:
• 6, SEQ ID NO:
• 7, SEQ ID NO:
• 8, SEQ ID NO:
• 9, SEQ ID NO:
• 10, SEQ ID NO:
• 11, SEQ ID NO:
• 12, SEQ ID NO:
• 13, SEQ ID NO:
• 15, and SEQ ID NO:
• 16;
• (b) identifying a DNA clone that hybridizes with the nucleic acid fragment of step (a);
• and
• (c) sequencing the nucleic acid fragment that comprises the DNA clone identified in step (b),
• wherein the sequenced nucleic acid fragment of step (b) encodes all or a substantial portion of an amino acid sequence encoding a nitrilase enzyme.

A method of obtaining a nucleic acid fragment encoding all or a substantial portion of a nitrilase enzyme, the method comprising:

• (a) synthesizing at least one oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of SEQ ID NO:
• 1, SEQ ID NO:
• 2, SEQ ID NO:
• 3, SEQ ID NO:
• 4, SEQ ID NO:
• 6, SEQ ID NO:
• 7, SEQ ID NO:
• 8, SEQ ID NO:
• 9, SEQ ID NO:
• 10, SEQ ID NO:
• 11, SEQ ID NO:
• 12, SEQ ID NO:
• 13, SEQ ID NO:
• 15, and SEQ ID NO:
• 16;
• and
• (b) amplifying an insert present in a cloning vector using the oligonucleotide primer of step (a), the amplified insert of step (b) encoding all or a substantial portion of an amino acid sequence encoding a nitrilase enzyme.

An improvement to the process for preparing five-membered ring lactams or six-membered ring lactams from aliphatic α,ω-dinitriles comprising:

• (a) contacting an aliphatic α,ω-dinitrile in an aqueous reaction mixture with an enzyme catalyst, whereby the aliphatic α,ω-dinitrile is converted to an ω-cyanocarboxylic acid ammonium salt;
• (b) contacting the aqueous product mixture resulting from step (a) with hydrogen and a hydrogenation catalyst, whereby the ω-cyanocarboxylic acid ammonium salt is converted directly to the corresponding lactam without isolation of the intermediate ω-cyanocarboxylic acid, ω-cyanocarboxylic acid ammonium salt, ω-aminocarboxylic acid, or ω-aminocarboxylic acid ammonium salt;
• and
• (c) recovering the lactam from the aqueous product mixture resulting from step (b),
• the improvement comprising in step (a) contacting an aliphatic α,ω-dinitrile in an aqueous reaction mixture with an enzyme catalyst selected from the group consisting of
• (1) E. coli SW91 having the designation ATCC PTA-1175;
• (2) E. coli DH5α:
• pnit4 having the designation ATCC PTA-1176;
• (3) E. coli SS1001 having the designation ATCC PTA-1177;
• and
• (4) E. coli SS1002 containing plasmid pnitex2, and
• (5) E. coli SS1011 containing plasmid pnitex2.

A method for using a native microbial gene encoding a protein characterized by a nitrilase activity on nitrilase-containing substrates to obtain a mutated microbial gene encoding a protein characterized by an increased specific nitrilase activity on nitrile-containing substrates and/or an increased stability of the nitrilase, one or both characteristics increased relative to that of the native microbial gene, the method comprising the steps of

• (i) contacting restriction endonucleases with a mixture of nucleotide sequences to yield a mixture of restriction fragments, the mixture of nucleotide sequences comprising
• a) a native microbial gene;
• b) a first population of nucleotide fragments which will hybridize with the nucleotide sequences of the native microbial gene of (i)(a);
• and
• c) a second population of nucleotide fragments which will not hybridize to the nucleotide sequences of the native microbial gene of (i)(a),
• (ii) denaturing the mixture of restriction fragments of step (i);
• (iii) incubating the denatured mixture of restriction fragments of step (ii) with a polymerase;
• and
• (iv) repeating steps (i), (ii), and (iii) a sufficient number of times to yield a mutated microbial gene encoding a protein characterized by an increased specific nitrilase activity on nitrile-containing substrates and/or an increased stability of the nitrilase, one or both characteristics increased relative to the nitrilase activity of the native microbial gene.