# US 2017/0283784 A1 · Improved nitrile hydratase

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- Number: US 2017/0283784 A1
- Document ID: US20170283784A1
- Status: Published application
- Kind: A1
- Application number: 15/316,328
- Filed: 2015-05-12
- Published: 2017-10-05
- Earliest claimed: 2014-06-06
- Assignees: Mitsubishi Chemical Corporation
- Applicants: Mitsubishi Chemical Corporation
- Inventors: Aya KITAHARA; Takanori AKIYAMA; Miki WAKAMATSU; Fumiaki WATANABE
- Web page: https://patentgrep.com/patent/US20170283784A1

## CPC

- C12N 9/88: Lyases (4.)
- C12N 5/10: Cells modified by introduction of foreign genetic material
- C12N 15/09: Recombinant DNA-technology
- C12P 13/02: Amides, e.g. chloramphenicol {or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes (peptides C12P21/00 or C07K)}
- C12Y 402/01084: Nitrile hydratase (4.2.1.84)
- 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

Provided is a novel improved nitrile hydratase with improved resistance to amide compounds under high temperatures. Specifically provided is a nitrile hydratase having at least one amino acid mutation selected from (a) to (d) below, in the amino acid sequence expressed in SEQ ID NO:50 (X1 to X27 represent independent arbitrarily-defined amino acid residuals). (a) X1 is valine or glycine (b) X9 is valine or threonine (c) X23 is an amino acid selected from a group consisting of isoleucine, leucine, methionine and threonine, (d) X24 is leucine.

## Claims

### Claim 1

A nitrile hydratase having at least one amino acid sequence represented by the following SEQ ID NO: 46, 47, 48 or 49 in an α subunit:
(a) SEQ ID NO: 46: X1X2X3X4X5X6RX7KAX8E
wherein X1 is an amino acid other than tyrosine;
X2, X3, X5, X6, X7 and X8 each independently are any amino acid residue; and
X4 is tyrosine;
(b) SEQ ID NO: 47: X9X10X11X12NX13X14X15X16X17X18X19X20X21CX22LC
wherein X9 is an amino acid other than serine; X10, X11, X12, X13, X14, X15, Xis, X17, X19, X20, X21 and X22 each independently are any amino acid residue; and X18 is histidine;
(c) SEQ ID NO: 48: X23WDSX25X26EX27RX28X29V
wherein X23 indicates an amino acid other than valine; X25, X27, X28 and X29 each independently are any amino acid residue; and X26 is serine;
(d) SEQ ID NO: 49: VX24DSX25X26EX27RX28X29V
wherein X24 is an amino acid other than tryptophan; X25, X27, X28 and X29 each independently are any amino acid residue; and X26 is serine.

### Claim 2 (depends on 1)

The nitrile hydratase according to claim 1, having at least the amino acid sequence represented by the following-SEQ ID NO: 47 in the α subunit:
(b) SEQ ID NO: 47: X9X10X11X12NX13X14X15X16X17X18X19X20X21CX22LC
wherein X9 is an amino acid other than serine; X10, X11, X12, X13, X14, X15, X16, X17, X19 and X20 each independently are any amino acid residue; X18 is histidine; X21 is valine; and X22 is threonine.

### Claim 3 (depends on 1)

The nitrile hydratase according to claim 1, having at least one amino acid sequence represented by the following SEQ ID NO: 46, 47, 48 or 49 in the α subunit:
(a) SEQ ID NO: 46: X1X2X3X4X5X6RX7KAX8E
wherein X1 is glycine or valine; X2, X3, X5, X6, X7 and X8 each independently are any amino acid residue; and X4 is tyrosine;
(b) SEQ ID NO: 47: X9X10X11X12NX13X14X15X16X17X18X19X20X21CX22LC
wherein X9 is valine or threonine; X10, X11, X12, X13, X14, X15, X16, X17, X19 and X20 each independently are any amino acid residue; X18 is histidine; X21 is valine; and X22 is threonine;
(c) SEQ ID NO: 48: X23WDSX25X26EX27RX28X29V
wherein X23 is selected from the group consisting of isoleucine, leucine, methionine, and threonine; X25, X27, X98 and X29 each independently are any amino acid residue; and X26 is serine;
(d) SEQ ID NO: 49: VX24DSX25X26EX27RX28X29V
wherein X24 is leucine X25, X27, X28 and X29 each independently are any amino acid residue; and X26 is serine.

### Claim 4 (depends on 1)

The nitrile hydratase according to claim 1, wherein
in SEQ ID NO: 46, X2 is threonine, X3 is glutamic acid, X4 is tyrosine, X5 is glutamic acid, X6 is alanine, X7 is threonine, and X8 is isoleucine,
in SEQ ID NO: 47, X10 is alanine, X11 is valine, X12 is phenylalanine, X13 is aspartic acid, X14 is serine, X15 is glutamine, X16 is threonine, X17 is histidine, X18 is histidine, X19 is valine and X20 is valine, and
in SEQ ID NO: 48 and 49, X25 is serine, X26 is serine, X27 is isoleucine, X28 is tyrosine and X29 is isoleucine.

### Claim 5 (depends on 1)

The nitrile hydratase according to claim 1, having an amino sequence represented by SEQ ID NO: 50.

### Claim 6

A nitrile hydratase having an amino acid sequence represented by SEQ ID NO: 50 in an α subunit, in which at least one amino acid mutation selected from the following (i) to (iv) is included:
(i) X1 is glycine or valine,
(ii) X9 is valine or threonine,
(iii) X23 is an amino acid selected from the group consisting of isoleucine, leucine, methionine, and threonine, and
(iv) X24 is leucine.

### Claim 7 (depends on 6)

The nitrile hydratase according claim 6, wherein X2 is threonine, X3 is glutamic acid, X4 is tyrosine, X5 is glutamic acid, X6 is alanine, X7 is threonine, X8 is isoleucine, X10 is alanine, X11 is valine, X12 is phenylalanine, X13 is aspartic acid, X14 is serine, X15 is glutamine, X16 is threonine, X17 is histidine, X18 is histidine, X19 is valine, X20 is valine, X25 is serine, X26 is serine, X27 is isoleucine, X28 is tyrosine, and X29 is isoleucine.

### Claim 8 (depends on 1)

The nitrile hydratase according to claim 1, wherein the nitrile hydratase is derived from Rhodococcus bacterium or Nocardia bacterium.

### Claim 9 (depends on 1)

DNA encoding the nitrile hydratase of claim 1, or DNA which hybridizes under stringent conditions with the DNA having a base sequence complementary to the above-mentioned DNA and encodes a protein having nitrile hydratase activity with enhanced resistance to amide compounds under high temperatures.

### Claim 10 (depends on 9)

A recombinant vector containing the DNA according to claim 9.

### Claim 11 (depends on 10)

A transformant containing the recombinant vector according to claim 10.

### Claim 12 (depends on 11)

A method for producing a nitrile hydratase, the method comprising culturing the transformant according to claim 11 and collecting the nitrile hydratase from the obtained culture.

### Claim 13 (depends on 1)

A method for producing an amide compound, the method comprising bringing a nitrile compound into contact with the nitrile hydratase according to claim 1.