Bacterial strain Rhodococcus aetherivorans VKM Ac-2610D producing nitrile hydratase, method of its cultivation and method for producing acrylamide

Patent metadata

Field	Value
Publication number	US-10138459-B2
Application number	US-201615337673-A
Country	US
Kind code	B2
Filing date	Oct 28, 2016
Priority date	Dec 27, 2012
Publication date	Nov 27, 2018
Grant date	Nov 27, 2018

Abstract

The invention relates to a bacterial strain belonging to the genus Rhodococcus which is a producer of a nitrile hydratase. The invention also relates to a method for producing acrylamide by hydration of acrylonitrile using a biomass of the bacterial strain and to a method of culturing the bacterial strain.

First claim

The invention claimed is:

1. A method of culturing a bacterial strain Rhodococcus aetherivorans VKM Ac-2610D, wherein cells of the strain are cultured using a nutrient medium comprising:
an aqueous phosphate buffer comprising sodium and potassium ions;
a source of carbon;
a source of nitrogen;
optionally an enzyme inducer;
optionally a cobalt salt;
a magnesium salt;
a zinc salt;
a calcium salt; and
a Fe(II) salt.

2. The method of claim 1 , wherein the cells of the bacterial strain are cultured until the growth of the cells of the bacterial strain enters the stationary phase or until a predetermined yield of cells is achieved.

3. The method of claim 1 , wherein the pH of the nutrient medium is 6.3-8.3.

4. The method of claim 1 , wherein the method is carried out at a temperature of 26-31° C.

5. The method of claim 1 , wherein the cells of the bacterial strain are cultured until the achievement of an optical density of 36-40.

6. The method of claim 1 , wherein the aqueous phosphate buffer comprises a phosphate salt selected from the group consisting of Na 2 HPO 4 , NaH 2 PO 4 , KH 2 PO 4 , K 2 HPO 4 , and any mixtures thereof.

7. The method of claim 1 , wherein the source of carbon is selected from the group consisting of glucose, cellobiose, fructose, galactose, maltose, mannose, sucrose, trehalose, ribose, glycerol, mannitol, sorbitol, salicin, inulin, citrate, pyruvate, succinate, fumarate, and any mixtures thereof.

8. The method of claim 1 , wherein the source of nitrogen is carbamide.

9. The method of claim 1 , wherein the cobalt salt is CoCl 2 , CoSO 4 , or a mixture thereof.

10. The method of claim 1 , wherein the magnesium salt is MgSO 4 .

11. The method of claim 1 , wherein the cells of the strain are seeded on a solid nutrient medium and cultivated for 24-48 hours, then the biomass is washed out, and a resulting suspension is used for inoculation of a first vessel comprising the nutrient medium, and the process is carried out during 24-48 hours with stirring to achieve an optical density of the suspension in the range of 2-16 units at a wavelength of 540 nm and a thickness of an optical layer of 5 mm; then the resulting suspension is used for inoculation of a second vessel having a volume which is 10-100 times larger than the volume of the first vessel, the second vessel comprising new nutrient medium, to achieve optical density of 0.1-0.3 in the second vessel; the process is continued for 48-120 hours with aeration until the achievement of an optical density of the suspension of 36-40 and a pH of 7.5-7.8; then the produced biomass is separated.

12. The method of claim 1 , wherein cells of the strain are seeded on a meat peptone agar slope and cultivated for 24-48 hours, then the biomass is washed out with a sterile physiological solution having a pH of 7.0-7.4, and a resulting suspension is used for inoculation of a first vessel comprising a nutrient medium of the following composition, g/l:

Na 2 HPO 4 · 12H 2 O
6.06

KH 2 PO 4
1.3

glucose
10.0-20.0

carbamide
2.0-6.0

MgSO 4 · 7H 2 O
1.0

ZnSO 4 · 7H 2 O
0.08-0.2 

CaCl 2 · H 2 O
0.2

CoCl 2 · 6H 2 O
0.01-0.02

FeSO 4 · 7H 2 O in a chelate complex form
 0.01-0.025

Distilled water
to 1 1

pH 7.0-7.4;

and the process is carried out during 24-48 hours at a temperature of 28-30° C. with a circular stirring at a rate of 140-160 rpm to achieve an optical density of the suspension in the range of 2-16 units at a wavelength of 540 nm and a thickness of an optical layer of 5 mm, then the resulting suspension is used for inoculation of a second vessel having a volume which is 10-100 times larger than a volume of the first vessel, the second vessel comprising a new nutrient medium of the following composition, g/l:

Na 2 HPO 4 · 12H 2 O
6.20

KH 2 PO 4
1.65

glucose
20.0-60.0

carbamide
10.0-24.0

MgSO 4 · 7H 2 O
0.8-1.0

ZnSO 4 · 7H 2 O
0.08-0.4 

a calcium salt
0.2-0.6

a cobalt salt
 0.04-0.085

FeSO 4 · 7H 2 O in a chelate complex form
0.025-0.05 

distilled water
to 1 1

pH 7.0-7.4;

to achieve optical density of 0.1-0.3 in the second vessel; the process is continued for 48-120 hours at a temperature of 26-31° C., an aeration of 0.5-1.0 volume of air/volume of medium per a minute until the achievement of an optical density of the suspension of 36-40 and a pH of 7.5-7.8; then the produced biomass is separated.

13. The method of claim 12 , wherein the nutrient medium in the second vessel comprises one of the following salts as a calcium salt:
CaHPO 4 .2H 2 O;
Ca(H 2 PO 4 ) 2 .2H 2 O,
Ca 3 (PO 4 ) 2 ;
CaCl 2 .2H 2 O;
CaCO 3 ;
CaSO 4 ;
Ca(C 3 H 5 O 3 ) 2 .3H 2 O; or
Ca(HOCH 2 (CHOH) 4 COO) 2 .H 2 O;
and one of following salts as a cobalt salt:
CoCl 2 .6H 2 O, or
CoSO 4 .7H 2 O.

14. The method of claim 1 , wherein the pH of the nutrient medium is 7.0-7.

Assignees

• Kemira Oyj

Inventors

• Kozulin Sergey Vladimirovich
• Kozulina Tatiana Nicolaevna
• Kozulin Alexey Sergeevich

Classifications

• C12R1/01

  Chemistry & Metallurgy · mapped topic

• C12R1/00

  Chemistry & Metallurgy · mapped topic

• C12P13/02

  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)} · CPC title

• C12Y402/01084

  Nitrile hydratase (4.2.1.84) · CPC title

• C12N1/20 Primary

  Bacteria; Culture media therefor · CPC title