# US 5,705,382 A · Method for preserving nitrile hydratase or nitrilase activity of microbial cells with inorganic salts

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- Number: US 5,705,382 A
- Document ID: US5705382A
- Status: Granted
- Kind: A
- Application number: 08/542,291
- Filed: 1995-10-12
- Granted: 1998-01-06
- Earliest claimed: 1994-10-14
- Assignees: Nitto Chemical Industry Co., Ltd.
- Inventors: Takakazu Endo; Toshiaki Doi; Koji Tamura; Yuji Hirata; Kouzo Murao
- Web page: https://patentgrep.com/patent/US5705382A

## CPC

- C12N 1/04: Preserving or maintaining viable microorganisms (immobilised microorganisms C12N11/00)
- C12N 11/14: Enzymes or microbial cells immobilised on or in an inorganic carrier
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Cells having enzyme activity, and the enzyme activity thereof, are preserved for a prolonged period of time, as a suspension of microbial cells or as a suspension of immobilized cells in particles, in an aqueous medium that is a neutral or weakly basic aqueous solution of inorganic salts, having a molarity ranging from 100 mM to the saturation concentration of the inorganic salts. Preferably, the microbial cells are cells containing the enzyme, nitrile hydratase or nitrilase, such as Gordona terrae or Rhodococcus rhodochrous, and the inorganic salts are phosphates, borates, sulfates, sulfites or hydrochlorides. The present invention provides an industrially useful method for preserving a large quantity of cells or immobilized cells in particles having nitrile hydratase or nitrilase enzyme activity for a prolonged period of time (e.g., 300 days) without cell lysis or enzyme deterioration even at room temperature. The present invention also renders possible a sharp reduction in labor and cooling cost, which are necessary in the conventional preservation process.

## Claims

### Claim 1

A method for stably preserving microbial cells having nitrile hydratase or nitrilase activity, and the nitrile hydratase or nitrilase activity thereof, for a prolonged period of time, comprising preserving said cells as a suspension of the microbial cells or as a suspension of cells immobilized in particles in an aqueous medium, wherein said aqueous medium consists of a neutral or weakly basic aqueous solution of inorganic salts, having a molarity ranging from 100 mM to the saturation concentration of said inorganic salts.

### Claim 2 (depends on 1)

The method according to claim 1, wherein said aqueous solution of inorganic salts is an aqueous solution of at least one salt selected from the group consisting of phosphates, borates, sulfates, sulfites and hydrochlorides.

### Claim 3 (depends on 1)

The method according to claim 1, wherein said inorganic salts are at least one selected from the group consisting of sodium salts, potassium salts and ammonium salts.

### Claim 4 (depends on 1)

The method according to claim 1, wherein said aqueous solution of inorganic salts has a pH value ranging from pH 7 to 10.

### Claim 5 (depends on 1)

The method according to claim 1, wherein said microbial cells are Gordona terrae MA-1 (FERM BP-4535).

### Claim 6 (depends on 1)

The method according to claim 1, wherein said microbial cells are Rhodococcus rhodochrous sp. HT40-6 (FERM BP-5231).

### Claim 7 (depends on 1)

The method according to claim 1, wherein said microbial cells are Rhodococcus rhodochrous J-1 (FERM BP-1478).

### Claim 8 (depends on 1)

The method according to claim 1, wherein said microbial cells are Rhodococcus rhodochrous ATCC 33278.

### Claim 9 (depends on 1)

The method according to claim 1, wherein said concentration of said cells immobilized in particles ranges from 0.1 to 30% by weight on a dry cell basis.

### Claim 10 (depends on 1)

The method according to claim 1, having a preservation temperature ranging from 0.degree. to 40.degree. C.

### Claim 11 (depends on 1)

The method according to claim 1, wherein said aqueous solution of inorganic salts comprises a phosphate buffer having a molarity ranging from 300-700 mM.

### Claim 12 (depends on 1)

The method according to claim 1, wherein said aqueous solution of inorganic salts comprises a phosphate buffer having a molarity of 50 mM and a sodium sulfate solution having a molarity of 300 mM.

### Claim 13 (depends on 1)

The method according to claim 1, wherein said cells immobilized in particles are suspended in an ammonium sulfate aqueous solution.