# US 7,608,445 B1 · Nitrilases, nucleic acids encoding them and methods for making and using them

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- Number: US 7,608,445 B1
- Document ID: US7608445B1
- Status: Granted
- Kind: B1
- Application number: 10/514,004
- Filed: 2003-05-15
- Granted: 2009-10-27
- Earliest claimed: 1999-12-29
- Assignees: Verenium Corporation
- Inventors: Grace Desantis; Jay M. Short; Mark J. Burk; Kelvin Wong; Robert Farwell; Kelly Chatman
- Web page: https://patentgrep.com/patent/US7608445B1

## CPC

- C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12P 7/42: Hydroxy-carboxylic acids
- C12P 13/04: Alpha- or beta- amino acids {(other amino acids C12P13/005)}
- C12P 41/006: {by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

The invention relates to nitrilases and to nucleic acids encoding the nitrilases. In addition methods of designing new nitrilases and method of use thereof are also provided. The nitrilases have increased activity and stability at increased pH and temperature.

## Claims

### Claim 1

An isolated, synthetic or recombinant nucleic acid, encoding a polypeptide having nitrilase activity, comprising
(a) a nucleic acid having a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete identity (100% identical) to SEQ ID NO:209; or
(b) a nucleic acid encoding a polypeptide comprising amino acids having a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete identity (100% identical) to SEQ ID NO:210; or
(c) a nucleic acid completely complementary to (a) or (b).

### Claim 2 (depends on 1)

A nucleic acid vector capable of replication in a host cell, wherein the vector comprises the nucleic acid of claim 1.

### Claim 3 (depends on 1)

A host cell comprising the nucleic acid of claim 1.

### Claim 4 (depends on 3)

A host organism comprising the host cell of claim 3.

### Claim 5 (depends on 1)

The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the nucleic acid is affixed to a solid support, wherein optionally the solid support is selected from the group of a gel, a resin, a polymer, a ceramic, a glass, a microelectrode and any combination thereof.

### Claim 6 (depends on 1)

A method for making a polypeptide having nitrilase activity, the method comprising
(a) introducing the nucleic acid of claim 1 encoding the polypeptide into a host cell under conditions that permit production of the polypeptide by the host cell, and
(b) recovering the recombinant polypeptide so produced.

### Claim 7 (depends on 1)

A kit comprising the nucleic acid of claim 1, or a fragment thereof encoding a polypeptide having nitrilase activity, and a buffer.

### Claim 8 (depends on 1)

The isolated, synthetic or recombinant nucleic acid of claim 1, having at least 95% sequence identity to SEQ ID NO:209.

### Claim 9 (depends on 1)

The isolated, synthetic or recombinant nucleic acid of claim 1, having at least 98% sequence identity to SEQ ID NO:209.

### Claim 10 (depends on 1)

The isolated, synthetic or recombinant nucleic acid of claim 1, having at least 99% sequence identity to SEQ ID NO:209.

### Claim 11 (depends on 1)

The isolated, synthetic or recombinant nucleic acid of claim 1, further consisting of one or more mutations, wherein:
nucleotides 163-165 of SEQ ID NO:209 are substituted with AAA, AAG, GGT, GGC, GGA, GGG, CAA, or CAG;
nucleotides 178-180 of SEQ ID NO:209 are substituted with GAA or GAG;
nucleotides 331-333 of SEQ ID NO:209 are substituted with TCT, TCC, TCA, TCG, AGT, or AGC;
nucleotides 568-570 of SEQ ID NO:209 are substituted with CAT, CAC, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, TCA, TAT, TAC, ATG or ACG;
nucleotides 571-573 of SEQ ID NO:209 are substituted with TTA, TTG, CTT, CTC, CTA, CTG, GTT, GTC, GTA, GTG, ATG, ACT, ACC, ACA, GAT, GAC, GGT, GGC, GGA, GGG, GAA, GAG, TAT, TAC, or ACG;
nucleotides 595-597 of SEQ ID NO:209 are substituted with GAA, GAG, TTA, TTG, CTT, CTC, CTA, or CTG;
nucleotides 664-666 of SEQ ID NO: 209 are substituted with TTA, TTG, CTT, CTC, CTA, or CTG; or any combination thereof.