# US 2021/0340518 A1 · Method for the production of acrylic acid or salts thereof

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- Number: US 2021/0340518 A1
- Document ID: US20210340518A1
- Status: Published application
- Kind: A1
- Application number: 17/286,328
- Filed: 2019-10-09
- Published: 2021-11-04
- Earliest claimed: 2018-10-18
- Assignees: BASF SE
- Applicants: BASF SE
- Inventors: Diego Ghislieri; Tobias Joachim Zimmermann; Stefan Seemayer; Michael Breuer; Doreen Schactschabel
- Web page: https://patentgrep.com/patent/US20210340518A1

## CPC

- C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12P 7/40: containing a carboxyl group {including Peroxycarboxylic acids}
- C12Y 305/05001: Nitrilase (3.5.5.1)
- 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)}
- C12Y: ENZYMES

## Abstract

Described herein are methods for the production of ammonium acrylate or salts thereof from acrylonitrile using nitrilase as a catalyst.

## Claims

### Claim 1

An isolated nitrilase capable of catalysing a reaction from (meth-) acrylonitrile to (meth-) ammonium acrylate in an aqueous medium comprising water, nitrilase and (meth-) acrylonitrile and/or (meth-) ammonium acrylate wherein the concentration of (meth-) ammonium acrylate in the aqueous medium after an incubation is at least 50% (w/w) and the concentration of (meth-) acrylonitrile is below 0.1% (w/w).

### Claim 2 (depends on 1)

The isolated nitrilase of claim 1 comprising a sequence selected from the group consisting of
a. An amino acid molecule of SEQ ID NO: 2, 4, 6 or 8,
b. An amino acid molecule having at least 55% identity to the amino acid molecule of SEQ ID NO: 2, 4, 6 or 8 or a functional fragment thereof,
c. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO: 1, 3, 5, or 7 or a functional fragment thereof,
d. An amino acid molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 1, 3, 5, or 7 or a functional fragment thereof, and
e. An amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 1, 3, 5, or 7 or a functional fragment thereof,
wherein the amino acid molecule as defined in b., d. and e. catalyses the reaction from (meth-) acrylonitrile to (meth-) ammonium acrylate in an aqueous medium.

### Claim 3

A process for producing (meth-) ammonium acrylate comprising the steps of
a. Providing an aqueous medium comprising water, one or more nitrilase and (meth-) acrylonitrile,
b. Incubating the aqueous medium and
c. Optionally isolating the (meth-) ammonium acrylate from the reaction mixture, wherein the one or more nitrilase is selected from the group consisting of
i. an amino acid molecule of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 18, 20, 22, 26, 28, 30, 32, 34, 38, 40, 42, 46, 48, 52, 54, 56, 60, 62, 64, 66 or 68 or a functional fragment thereof,
ii. an amino acid molecule having at least 55% identity to the amino acid molecule of SEQ ID NO: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 18, 20, 22, 26, 28, 30, 32, 34, 38, 40, 42, 46, 48, 52, 54, 56, 60, 62, 64, 66 or 68 or a functional fragment thereof,
iii. an amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 37, 39, 41, 45, 47, 51, 53, 55, 59, 61, 63, 65 or 67 or a functional fragment thereof,
iv. an amino acid molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 37, 39, 41, 45, 47, 51, 53, 55, 59, 61, 63, 65 or 67 or a functional fragment thereof, and
v. an amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 37, 39, 41, 45, 47, 51, 53, 55, 59, 61, 63, 65 or 67 or a functional fragment thereof,
wherein the amino acid molecule as defined in ii., iv. and v. has an activity of converting (meth-) acrylonitrile to (meth-) ammonium acrylate.

### Claim 4 (depends on 3)

The process of claim 3 wherein the (meth-) acrylonitrile is continuously added to the aqueous medium until 10 min before end of the incubation time thereby keeping the (meth-) acrylonitrile concentration at about 1-2% w/w.

### Claim 5 (depends on 3)

The process of claim 3 wherein the aqueous medium is incubated for at least 2 h up to 48 h.

### Claim 6 (depends on 3)

The process of claim 3 wherein the aqueous medium is incubated between 15 and 50° C.

### Claim 7 (depends on 3)

The process of claim 3 wherein the nitrilase is produced by fermentation.

### Claim 8

An isolated nitrilase comprising a sequence selected from the group consisting of
a. an amino acid molecule of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 18, 20, 22, 26, 28, 30, 32, 34, 38, 40, 42, 46, 48, 52, 54, 56, 60, 62, 64, 66 or 68 or a functional fragment thereof,
b. an amino acid molecule having at least 55% identity to the amino acid molecule of SEQ ID NO: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 18, 20, 22, 26, 28, 30, 32, 34, 38, 40, 42, 46, 48, 52, 54, 56, 60, 62, 64, 66 or 68 or a functional fragment thereof,
c. an amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 37, 39, 41, 45, 47, 51, 53, 55, 59, 61, 63, 65 or 67 or a functional fragment thereof,
d. an amino acid molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 37, 39, 41, 45, 47, 51, 53, 55, 59, 61, 63, 65 or 67 or a functional fragment thereof, and
e. an amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 37, 39, 41, 45, 47, 51, 53, 55, 59, 61, 63, 65 or 67 or a functional fragment thereof,
wherein the amino acid molecule as defined in b., d. and e. catalyses a reaction from (meth-) acrylonitrile to (meth-) ammonium acrylate in an aqueous medium.

### Claim 9

A recombinant construct comprising a nitrilase wherein the nitrilase is selected from the group consisting of
i. an amino acid molecule of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 18, 20, 22, 26, 28, 30, 32, 34, 38, 40, 42, 46, 48, 52, 54, 56, 60, 62, 64, 66 or 68 or a functional fragment thereof,
ii. an amino acid molecule having at least 55% identity to the amino acid molecule of SEQ ID NO: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 18, 20, 22, 26, 28, 30, 32, 34, 38, 40, 42, 46, 48, 52, 54, 56, 60, 62, 64, 66 or 68 or a functional fragment thereof,
iii. an amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 37, 39, 41, 45, 47, 51, 53, 55, 59, 61, 63, 65 or 67 or a functional fragment thereof,
iv. an amino acid molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 37, 39, 41, 45, 47, 51, 53, 55, 59, 61, 63, 65 or 67 or a functional fragment thereof, and
v. an amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 37, 39, 41, 45, 47, 51, 53, 55, 59, 61, 63, 65 or 67 or a functional fragment thereof,
wherein the amino acid molecule as defined in ii., iv. and v. catalyses a reaction from (meth-) acrylonitrile to (meth-) ammonium acrylate in an aqueous medium.

### Claim 10 (depends on 9)

The recombinant construct of claim 9, wherein the nitrilase is functionally linked to a heterologous promoter.

### Claim 11 (depends on 9)

A recombinant vector comprising the recombinant construct of claim 9.

### Claim 12 (depends on 9)

A recombinant microorganism comprising the recombinant construct of claim 9.

### Claim 13 (depends on 12)

The recombinant microorganism of claim 12 wherein the microorganism is Rhodococcus rhodocrous, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, or Pichia pastoris.

### Claim 14 (depends on 8)

A microorganism of the genus Cupriavidus basilensis, Flavihumibacter solisilvae, Acidovorax facilis 72W, Pseudomonas sp. RIT357, Nocardia brasiliensis NBRC 14402, Pseudomonas fluorscens, Agrobacterium rubi, Candidatus Dadabacteria bacterium CSP1-2, Tepidicaulis marinus, Synechococcus sp. CC9605, Aquimarina atlantica, Arthrobacter sp., Sphingomonas wittichii RW1, Pseudomonas mandelii JR-1, Salinisphaera shabanensis E1L3A, Smithella sp. SDB, Bradyrhizobium diazoefficiens, Actinobacteria bacterium RBG_13_55_18, Rhizobium sp. YK2 or Bacterium YEK0313 expressing the nitrilase of claim 8.

### Claim 15 (depends on 12)

A method for producing a nitrilase, comprising the steps of
a) providing a recombinant microorganism according to claim 12, and
b) cultivating the microorganism under conditions allowing for the expression of a nitrilase gene.

### Claim 16

A composition comprising water, a nitrilase, (meth-) acrylonitrile and/or (meth-) ammonium acrylate wherein the nitrilase is selected from the group consisting of
i. an amino acid molecule of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 18, 20, 22, 26, 28, 30, 32, 34, 38, 40, 42, 46, 48, 52, 54, 56, 60, 62, 64, 66 or 68 or a functional fragment thereof,
ii. an amino acid molecule having at least 55% identity to the amino acid molecule of SEQ ID NO: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 18, 20, 22, 26, 28, 30, 32, 34, 38, 40, 42, 46, 48, 52, 54, 56, 60, 62, 64, 66 or 68 or a functional fragment thereof,
iii. an amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 37, 39, 41, 45, 47, 51, 53, 55, 59, 61, 63, 65 or 67 or a functional fragment thereof,
iv. an amino acid molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 37, 39, 41, 45, 47, 51, 53, 55, 59, 61, 63, 65 or 67 or a functional fragment thereof, and
v. an amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 37, 39, 41, 45, 47, 51, 53, 55, 59, 61, 63, 65 or 67 or a functional fragment thereof,
wherein the amino acid molecule as defined in ii., iv. and v. catalyses a reaction from (meth-) acrylonitrile to (meth-) ammonium acrylate in an aqueous medium.

### Claim 17 (depends on 11)

A recombinant microorganism comprising the recombinant vector of claim 11.

### Claim 18 (depends on 17)

The recombinant microorganism of claim 17 wherein the microorganism is Rhodococcus rhodocrous, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, or Pichia pastoris.

### Claim 19 (depends on 17)

A method for producing a nitrilase, comprising the steps of
a) providing a recombinant microorganism according to claim 17, and
b) cultivating the microorganism under conditions allowing for the expression of a nitrilase gene.