Nitrilases, nucleic acids encoding them and methods for making and using them

1. An isolated, a synthetic, or a recombinant polynucleotide comprising a nucleic acid sequence identical to SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 31, wherein the nucleic acid sequence is operably linked to a heterologous promoter.

2. A recombinant expression cassette, a vector, or a cloning vehicle comprising the polynucleotide of claim 1 .

3. A method for hydrolyzing a nitrile to a carboxylic acid comprising contacting the molecule with the polypeptide encoded by the nucleic acid of claim 1 .

4. A method for hydrolyzing a cyanohydrin moiety or an aminonitrile moiety of a molecule, the method comprising contacting the molecule with the polypeptide encoded by the nucleic acid of claim 1 .

5. A method for making a chiral alpha-hydroxy acid molecule or a chiral amino acid molecule, the method comprising admixing a molecule having a cyanohydrin moiety or an aminonitrile moiety with the polypeptide encoded by the nucleic acid of claim 1 .

6. A method for making a composition or an intermediate thereof, the method comprising admixing a precursor of the composition or intermediate, wherein the precursor comprises a cyanohydrin moiety or an aminonitrile moiety, with the polypeptide encoded by the nucleic acid of claim 1 , hydrolyzing the cyanohydrin or the aminonitrile moiety in the precursor thereby making the composition or the intermediate thereof.

7. A method for making an (R)-ethyl 4-cyano-3-hydroxybutyric acid, the method comprising contacting a hydroxyglutaryl nitrile with the polypeptide encoded by the nucleic acid of claim 1 , or a fragment thereof encoding a polypeptide having nitrilase activity, that selectively produces an (R)-enantiomer, so as to make (R)-ethyl 4-cyano-3-hydroxybutyric acid.

8. A method for making an (S)-ethyl 4-cyano-3-hydroxybutyric acid, the method comprising contacting a hydroxyglutaryl nitrile with the polypeptide of claim 1 or peptidomimetic thereof having nitrilase activity that selectively produces an (S)-enantiomer, so as to make (S)-ethyl 4-cyano-3-hydroxybutyric acid.

9. A method for making an (R)-mandelic acid, the method comprising admixing a mandelonitrile with the polypeptide encoded by the nucleic acid of claim 1 , or peptidomimetic thereof having nitrilase activity.

10. A method for making an (S)-mandelic acid, the method comprising admixing a mandelonitrile with the polypeptide encoded by the nucleic acid of claim 1 , or peptidomimetic thereof having nitrilase activity.

11. A method for making an (S)-phenyl lactic acid derivative or an (R)-phenyl lactic acid derivative, the method comprising admixing a phenyllactocyanonitrile with the polypeptide encoded by the nucleic acid of claim 1 , or peptidomimetic thereof having nitrilase activity, or any active fragment or peptidomimetic thereof that selectively produces an (S)-enantiomer or an (R)-enantiomer, thereby producing an (S)-phenyl lactic acid derivative or an (R)-phenyl lactic acid derivative.

12. A method for generating a nucleic acid variant encoding a polypeptide having nitrilase activity, wherein the variant has an altered biological activity from that which naturally occurs, the method comprising

(a) modifying the nucleic acid of claim 1 by

(i) substituting one or more nucleotides for a different nucleotide, wherein the nucleotide comprises a natural or non-natural nucleotide;

(ii) deleting one or more nucleotides,

(iii) adding one or more nucleotides, or

(iv) any combination thereof.

13. A method for making a polynucleotide from two or more nucleic acids, the method comprising:

(a) identifying regions of identity and regions of diversity between two or more nucleic acids, wherein at least one of the nucleic acids comprises the nucleic acid of claim 1 ;

(b) providing a set of oligonucleotides which correspond in sequence to at least two of the two or more nucleic acids; and,

(c) extending the oligonucleotides with a polymerase, thereby making the polynucleotide.

14. A method for modifying a molecule comprising:

(a) mixing a polypeptide encoded by the nucleic acid of claim 1 , or peptidomimetic thereof having nitrilase activity, with a starting molecule to produce a reaction mixture;

(b) reacting the starting molecule with the polypeptide to produce the modified molecule.

15. A method for identifying a modified compound comprising:

(a) admixing a polypeptide encoded by the nucleic acid of claim 1 , or peptidomimetic thereof having nitrilase activity, with a starting compound to produce a reaction mixture and thereafter a library of modified starting compounds;

(b) testing the library to determine whether a modified starting compound is present within the library which exhibits a desired activity;

(c) identifying the modified compound exhibiting the desired activity.

16. An assay for identifying a functional fragment of a polypeptide which comprises:

(a) obtaining a fragment of the polypeptide encoded by the nucleic acid of claim 1 ;

(b) contacting at least one fragment from step (a) with a substrate having a cyanohydrin moiety or an aminonitrile moiety under reaction conditions suitable for nitrilase activity;

(c) measuring the amount of reaction product produced by each at least one fragment from step (b); and

(d) identifying the at least one fragment which is capable of producing a nitrilase reaction product; thereby identifying a functional fragment of the polypeptide.