EA032896B1 - Method for cultivating the strain rhodococcus rhodochrous m33 - Google Patents

Abstract

The invention relates to bio technologies and concerns a laboratory and commercial method for cultivating the Rhodococcus rhodochrous M33, a producer of nitrile hydratase. The cultivating method comprises addition of sources of asparagine acid anions and metal ions, i.e. iron, magnesium and cobalt ions, to the culture medium. The invention also includes a medium for cultivating the Rhodococcus rhodochrous M33 containing a source of asparagine acid anions and sources of metal ions of iron, magnesium and cobalt. The invention makes it possible to commercially produce a biomass of the Rhodococcus rhodochrous M33 producing strains having high nitrile hydratase activity.

Description

The invention relates to the field of biotechnology and relates to a laboratory and industrial method of cultivating a strain of Rhodococcus rhodochrous M33.

State of the art

Currently, polymers and copolymers of acrylamide are widely used. However, the global demand for these products is not fully satisfied and is increasing annually by 4-5%. Unlike traditional chemical methods, the biocatalytic method for the production of acrylamide has several important advantages: high specificity and selectivity, one-stage, low energy consumption, the absence of synthesis by-products, technological effluents, and hazardous waste. Technologies for the industrial production of acrylamide using biocatalysts based on strains producing nitrile hydratase enzyme are increasingly being applied. The expansion of studies of bacterial strains metabolizing nitriles is due to the need to improve the method of acrylamide production, as well as the prospect of using microorganisms to obtain a number of other commercially significant compounds.

Along with the search for new strains producing the nitrile hydratase enzyme, much attention is paid to the method of cultivation of these microorganisms. This is necessary in order to be able to obtain catalytically active biomass on an industrial scale. To increase the yield of enzymatic activity per unit volume of culture fluid, two main methods are usually used:

1) activation of the synthesis of the enzyme by a bacterial cell or, in other words, an increase in the specific enzymatic activity (U / mg dry biomass);

2) increasing the yield of active cells (active biomass) per unit volume of culture fluid.

GB2087926 describes a method for culturing a strain of Corynebacterium N-774 (registration number FERM 4446 at the Fermentation Research Institute). The strain is cultivated in a nutrient medium containing water-soluble iron compounds in concentrations of at least 0.2 mg / l, together with 1% casein hydrolyzate (casamic acids). The disadvantages of this method are the high cost of casein hydrolysates and the resulting low nitrile hydratase activity of the biomass (specific activity = 53.3 U / mg, dry biomass = 5.66 mg / ml, total activity = 301.7 U / ml).

It is known that nitriles and amides of organic acids, in particular propionic and isobutyric, induce the synthesis of nitrile hydratase. This can be concluded on the basis of the data of US4555487, which shows that the addition of substances known as nitrile hydratase synthesis inducers to the culture medium of the strain Pseudomonas chlororaphis B 23 (FERM BP-187) and the strain Pseudomonas sp. PS 1 (FERM BP-188), makes it possible to obtain biomass with nitrile hydratase activity (dry biomass - 5.36 g / l, specific activity - 62.65 U / mg, total activity 335.8 U / ml).

The disadvantages inherent in this method are associated with the use of expensive inducers and obtaining cells with low nitrile hydratase activity.

The culture medium for incubation of the strain Pseudomonas chlororaphis B 23 (FERM BP-187) and the strain Pseudomonas sp. PS 1 (FERM BP-188) contains not only inducers, but also one or more α-amino acids, such as cysteine, alanine, valine, methionine, etc., in concentrations from 0.1 to 10.0 g / l as agents that increase enzymatic activity. However, the specific activity of the biomass obtained by this method according to patent EP0115781 does not exceed 105.7 U / mg, and the total activity does not exceed 428.1 U / ml.

Higher nitrile hydratase activity was achieved by incubating the strain Rhodococcus rhodochrous M33 (patent DE4480132). In the description of the patent it is indicated that when cultivating the strain on a synthetic nutrient medium containing glucose at a concentration of 5 g / l, it is quite possible to obtain cells with nitrile hydratase activity of up to 200 U / mg and a total activity of up to 360 U / ml. An important advantage of the proposed method is the simplicity of the nutrient medium, which does not contain any expensive components. However, an increase in glucose concentration to 20 g / l leads to an increase in total activity to a value not exceeding 1368 U / ml.

RU2223316 describes a method for culturing a strain of Rhodococcus ruber GT capable of producing nitrile hydratase with an activity of 330 U / mg. Cultivation is carried out on a medium containing Fe ^{2+ ions} and acetonitrile as an inductor. The disadvantage of this method is the inability to obtain more or less significant biomass yields.

Patent RU2403280 describes a method for culturing a strain of Rhodococcus rhodochrous NCIMB 41164 showing nitrile hydratase activity of 323 U / mg at 30 ° C. The medium for its cultivation contains acetonitrile - as an inducer and yeast extract - as a source of growth factors. However, this method of cultivation is intended only for laboratory conditions and is not suitable for industry.

EP0362829 describes a method for cultivating a strain of Rhodococcus rhodochrous J1 (FERM BP-1478). When culturing cells of this strain on a nutrient medium containing compounds

- 1 032896 of bivalent cobalt and urea at a concentration of 15 g / l, it was possible to obtain a biomass showing a specific activity of 578 U / mg. To increase the yield of cells in the nutrient medium, peptone and yeast extract were added. Nevertheless, the yield of dry biomass was only 4.3 g / l, and the achieved total activity did not exceed 2480 U / ml. Scaling the cultivation process to production conditions increased the yield of cells to 28 g / l, but this was accompanied by a decrease in specific nitrile hydratase activity to 76 U / mg and a decrease in total activity to 2100 U / ml (see Yamada H., Kobayashi M., Nitrile hydratase and Application to Industrial Production of Acrylamide, Biosci. Biotech. Biochem., 60 (9), 1391-1400, 1996).

Another method of cultivating the strain Rhodococcus rhodochrous M33 is characterized in that the cells of the strain are cultured in a 5-liter fermenter on a fully synthetic medium consisting of KH ₂ PO ₄ , K ₂ HPO ₄ , FeSO ₄ -7H ₂ O, EDTA-2 No. 1. MgSO ₄ -7H ₂ O, NaCl, CoCl ₂ -6H ₂ O, urea and glucose. By continuously regulating the supply of a 40% glucose solution and three periodic additions of CoCl ₂ -6H ₂ O (0.01 g / l each), a cell yield of 24 g / l and a specific nitrile hydratase activity of 120 U / mg were achieved. However, the total activity after a relatively long fermentation time (115 hours) did not exceed 2880 U / ml (see Kim BY, Kim JC, Lee HH., Hyun HH., Fed-batch Fermentation for Production of Nitrile Hydratase by Rhodococcus rhodochrous M33, Biotechnol Bioprocess Eng., 6: 1117, 2001).

A method that meets industry requirements is described in patent RU2340667. The strain Rhodococcus rhodochrous M33 was cultured in a 15 m ³ fermenter in a medium containing corn extract as a source of growth factors, urea and CoCl ₂ -6H ₂ O as inducers and glucose as a carbon source. After 24 to 36 hours after the start of cultivation and consumption of all glucose, 6 g / L of urea, 3 g / L of sodium acetate and 0.03 g / L of CoCl ₂ -6H ₂ O were added to the medium; The pH of the culture medium was maintained at 7.9 by the addition of an aqueous solution of acetic acid. After 72 hours of fermentation, 18.8 g / L dry biomass with a nitrile hydratase activity of 290 U / mg was obtained. The total activity was 5452 U / ml. However, due to the increasing need for biocatalysts hydrolyzing acrylate nitrile to acrylamide, the problem of increasing the nitrile hydratase activity of the strains remains relevant

Disclosure of Invention

The objective of the present invention is to develop an effective industrial method for producing a large amount of biomass of the strain Rhodococcus rhodochrous M33 with high total nitrile hydratase activity.

The problem is solved by developing a method for the cultivation of Rhodococcus rhodochrous M33, a producer of nitrile hydratase, characterized in that the cultivation medium used includes anions of aspartic acid, as well as metal ions - iron, magnesium and cobalt.

In some embodiments of the invention, the sources of metal ions are metal aspartates and / or inorganic metal salts.

In some particular embodiments, the source of at least one of the iron, magnesium, or cobalt ions is, respectively, iron (II) aspartate, iron (III) aspartate, magnesium (II) aspartate, or cobalt (II) aspartate.

In some particular embodiments, the inorganic metal salt is sulfate, chloride, and / or hydrates thereof.

In some particular embodiments, the inorganic salt is FeSO ₄ , MgSO 4, CoCl 2, and / or their hydrates.

In some embodiments of the invention, the source of aspartic acid anions is aspartic acid, iron aspartate, magnesium aspartate, and / or cobalt aspartate.

In some embodiments of the invention, the source of anions of aspartic acid is aspartic acid, and the source of at least one of the metal ions is its inorganic salt.

In some embodiments of the invention, the total concentration of anions of aspartic acid in the medium is 0.076-3.0 g / L.

In some embodiments of the invention, in the course of cultivation, after 30-40 hours from the start of cultivation, glucose is additionally added to the medium so that the total amount of glucose in the medium does not exceed 120.0 g / l.

In some embodiments of the invention, sodium acetate is additionally added to the culture medium so that the total amount of sodium acetate added does not exceed 6.0 g / L.

In some particular embodiments of the invention, the pH of the medium is maintained at a predetermined level by an acetic acid solution.

The problem is also solved by developing a culture medium for the cultivation of Rhodococcus rhodochrous M33, a producer of nitrile hydratase, characterized in that the medium includes a source of aspartic acid anions, as well as sources of metal ions - iron, magnesium and cobalt.

In some embodiments of the invention, in a culture medium, metal ion sources are provided

- 2 032896 are metal aspartates and / or inorganic metal salts.

In some particular embodiments of the invention, in a culture medium, the source of at least one of the iron, magnesium, or cobalt ions is, respectively, iron (II) aspartate, iron (III) aspartate, magnesium (II) aspartate, or cobalt (II) aspartate .

In some particular embodiments of the invention, in the culture medium, the inorganic salt is sulfate, chloride and / or hydrates thereof.

In some embodiments of the invention in a culture medium, the inorganic salt is FeSO ₄ , CoCl _{2 —} MgSO ₄ and / or their hydrates.

In some embodiments of the invention, in a culture medium, the source of aspartic acid anions is aspartic acid, iron aspartate, magnesium aspartate, and / or cobalt aspartate.

In some embodiments of the invention, in the culture medium, the source of aspartic acid anions is aspartic acid, and the source of at least one of the metal ions is its inorganic salt.

In some embodiments of the invention, in the culture medium, the total concentration of anions of aspartic acid is 0.076-3.0 g / L.

In some particular embodiments of the invention, the culture medium comprises, g / l:

Na ₂ HPO ₄ -12H ₂ O - 2.2 - 13.4;

KN ₂ RO ₄ - 0.5 - 3.1;

FeSO ₄ -7H ₂ O- 0.01-0.3;

EDTA Na (sodium salt of ethylenediaminetetraacetic acid) - 0.02 - 0.6;

CoCl ₂ -6H ₂ O - 0.01 - 0.08;

MgSO ₄ -7H ₂ O - 0.05 - 2.5;

urea - 4.0 - 20.0;

glucose -12.0 - 120.0;

aspartic acid - 0.076 - 3.0;

water is the rest.

In some other particular embodiments of the invention, the culture medium comprises, g / l:

Na ₂ HPO ₄ 12H ₂ O - 2.2 -13.4;

KH ₂ PO ₄ -0.5-3.1;

iron aspartate (H) - 0.011-0.33;

EDTA Na - 0.02 - 0.6;

cobalt aspartate (P) - 0.014 - 0.11;

magnesium aspartate (P) - 0.06 - 2.9;

urea - 4.0 - 20.0;

glucose-12.0 - 120.0;

water is the rest.

As a result of the invention, the following technical results are achieved: the yield of the active biomass of Rhodococcus rhodochrous M33 and the nitrile hydratase activity of Rhodococcus rhodochrous M33 are increased;

a medium was developed for Rhodococcus rhodochrous M33, which allows cultivating strain producer without introducing growth factors into the medium;

a method of industrial cultivation of Rhodococcus rhodochrous M33 has been developed, providing high enzymatic activity;

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to an effective industrial method for producing biomass of Rhodococcus rhodochrous M33 with high total nitrile hydratase activity.

The claimed method consists in the development of cultivation conditions, as well as the creation of a culture medium without the use of growth factors and expensive inductors that provide increased biomass yield and high total nitrile hydratase activity of Rhodococcus rhodochrous M33.

To obtain the biomass of the strain Rhodococcus rhodochrous M33, it is necessary to develop a culture medium that fully meets the growth needs of the cells. In the research process, preference was given to a synthetic medium that does not contain expensive inductors and sources of growth factors, since the sources of growth factors differ in composition depending on the manufacturer, and it is almost impossible to determine the exact composition.

The method consists in introducing into the culture medium of the nitrile hydratase producer anions (residues) of aspartic acid (aspartate ions) and metal ions - iron, magnesium and cobalt.

Sources of metal ions are metal aspartates and / or inorganic metal salts.

In private embodiments of the invention, the source of at least one of the ions of iron, magnesium or cobalt is, respectively, iron (II) aspartate, iron (III) aspartate, aspartate

- 3 032896 magnesium (II) or cobalt (II) aspartate. In other embodiments, the source of at least one of the ions of iron, magnesium, or cobalt is the corresponding inorganic salt. Inorganic metal salts may be, in particular, sulfates, chlorides or their hydrates. Examples of such inorganic salts are FeSO ₄ , MgSO ₄ , CoCl ₂ , as well as their hydrates, for example: FeSO4-7 ^ O, FeSO4-4H2O, FeSO4-H2O, CoCM ^ O, CoC ^^ O, CoCM ^ O, CoC ^^ O, CoCl2-H2O, MgSO4-12H2O, MgSO4-7 ^ O, MgSO4-6 ^ O, MgSO4-5H2O, MgSO4-4H2O, MgSO4-3H2O, MgSO ₄ -2H ₂ O, MgSO ₄ -H ₂ O. In order to implement the invention, it is possible to use other inorganic salts of iron, magnesium and cobalt ions, in addition to the above, provided that these salts will not adversely affect the growth of cells of the strain Rhodococcus rhodochrous M33 and the synthesis of nitrile hydratase. For example, FeCl ₂ , MgCl ₂ and their hydrates, for example, FeCl ₂ -6H ₂ O; FeCl ₂ -4H ₂ O; FeCl ₂ -2H ₂ O, FeCl2-H2O, MgCl2-6H2O, MgC ^^ O.

The invention also encompasses variants in which metal aspartates and their inorganic salts, in various combinations, are simultaneously sources of metal ions in the culture medium. For example, iron aspartate and inorganic cobalt and magnesium salts are introduced into the culture medium; cobalt aspartate and inorganic salts of iron and magnesium; magnesium aspartate and inorganic salts of cobalt and iron. It is also possible to include in the cultivation medium both aspartate and an inorganic salt of one of the necessary sources of metal ions (iron, cobalt or magnesium), for example, iron in the form of aspartate and iron in the form of chloride; or, for example, magnesium in the form of aspartate together with magnesium in the form of chloride and in the form of sulfate. Sources of metal ions are introduced in the form of iron (II), iron (III), magnesium (II) or cobalt (II). Thus, the invention provides the possibility of using iron (P) and iron (III), as well as the presence of all these metals in the culture medium in the form of metal cations Co ²⁺ ; Fe ²⁺ and Mg ²⁺ .

The source of aspartic acid anions in the culture medium is aspartic acid, iron aspartate, magnesium aspartate and / or cobalt aspartate. In some cases, the source of anions of aspartic acid is aspartic acid, and the source of at least one of the metal ions is inorganic salts. It is also possible to simultaneously add metal aspartates (iron, cobalt and magnesium), aspartic acid and inorganic metal salts (iron, cobalt and magnesium) to the culture medium. The total concentration of anions of aspartic acid in the medium is 0.076-3.0 g / l.

When iron, magnesium and cobalt ions are introduced in the form of salts of aspartic acid and / or inorganic salts with the addition of an anion of aspartic acid, the anion of aspartic acid acts as a transporter (carrier) of ions through the cell wall.

Other essential components of the culture medium are carbon sources. As sources of carbon and energy can be used: glucose, acetic acid, sodium acetate. Urea was used as a nitrogen source and an inducer of nitrile hydratase synthesis. The use of these components in a culture medium is known in the art, for example, Kim B-Y, Kim J-C, Lee H-H., Hyun H-H., Fed-batch Fermentation for Production of Nitrile Hydratase by Rhodococcus rhodochrous M33, // Biotechnol. Bioprocess Eng., 6: 11-17, 2001; RU2340667. Carbon sources are introduced into the culture medium in one or two parts (fractionally) to a final concentration with a certain time interval. In particular, in the course of cultivation, after 30-40 hours from the start of cultivation, glucose is additionally added to the medium so that the total amount of glucose in the medium does not exceed 120.0 g / l. It is also contemplated within the framework of the present invention to use a two-component carbon source application scheme involving the use of a first carbon source in a first growth step and introducing a second carbon source after consuming the first carbon source. In particular, sodium acetate is additionally added to the culture medium so that the total amount of sodium acetate introduced does not exceed 6.0 g / l. When sodium acetate is added, the pH of the medium is maintained at a predetermined level by an acetic acid solution.

A phosphate buffer is used as the basis for the cultivation medium, which satisfies the phosphorus needs of the strain cells and maintains the pH value in a given range.

An example of a culture medium for Rhodococcus rhodochrous M33 according to the invention can be a medium of the following composition (g / l):

Na ₂ HPO ₄ -12H ₂ O 2.2-13.4; KN ₂ RO ₄ 0.5-3.1; FeSO ₄ -7H ₂ O 0.01-0.3; EDTA Na 0.02 - 0.6; CoCI ₂ -6H ₂ 0 0.01-0.08; MgSO ₄ -7H ₂ O 0.05-2.5; Urea 4.0 to 20.0; Glucose 12.0 - 120.0; Aspartic acid 0.076-3.0; Water is the rest.

- 4 032896

Another example of a culture medium may be, for example, a medium of the following composition (g / l):

Na ₂ HPO ₄ -12H ₂ O 2.2-13.4;

KH2RO4 0.5-3.1;

Aspartate of iron (II) 0.011-0.33;

EDTA Na 0.02 - 0.6;

Aspartate of cobalt (II) 0.014 - 0.11;

Aspartate of magnesium (II) 0.06 - 2.9;

Urea 4.0 - 20.0;

Glucose 12.0-120.0;

Water is the rest.

The proposed cultivation method can be used, preferably, for the cultivation of microorganisms from the genus Rhodococcus, more preferably for microorganisms of the species Rhodococcus rhodochrous, most preferably for the strain Rhodococcus rhodochrous deposited in the All-Russian collection of microorganisms under the number VKM Ac-1515D, hereinafter referred to as Rhodococcus rhodochrous .

The method is as follows.

In one embodiment of the invention, phosphate buffer at a concentration of 10-60 mM was taken as the basis of the medium, which maintained the pH value in the physiological range of 5.0-9.0. Typically, cultivation was carried out in 35.3 mm phosphate buffer.

The cultivation medium (abbreviated hereinafter as MS), containing a source of metal ions in the form of inorganic salts of iron, cobalt and magnesium, had the following composition (g / l):

1. Na ₂ HPO ₄ -12H ₂ O 2.2-13.4; 2. KN ₂ RO ₄ 0.5-3.1; 3. FeSO ₄ -7H ₂ O 0.01-0.3; 4. EDTA Na 0.02 - 0.6; 5. CoCI ₂ -6H ₂ O 0.01-0.08; 6. MgSO ₄ -7H ₂ O 0.05-2.5; 7. Urea 4.0 to 20.0; 8. Gpukoza 12.0-120.0; 9. Sodium Acetate 2.0-6.0 (when growing in two-component

scheme);

10. Water - the rest.

According to the invention, for the chelation of iron, cobalt and magnesium ions, aspartic acid at a concentration of 0.076-3.0 g / l was introduced into the MS medium.

The cultivation medium (abbreviated hereinafter as MS-1) containing sources of metal ions - iron, cobalt and magnesium in the form of salts of aspartic acid, had the following composition (g / l):

1. Na ₂ HPO ₄ -12H ₂ O 2.2-13.4; 2. KN ₂ RO ₄ 0.5-3.1; 3. Aspartate of iron (II) 0.011-0.33; 4. EDTA Na 0.02 - 0.6; 5. Cobalt Aspartate (II) 0.014-0.11; 6. Magnesium Aspartate (II) 0.06-2.9; 7. Urea 4.0 to 20.0; 8. Glucose 12.0-120.0; 9. Sodium Acetate 2.0-6.0 (p

cultivation according to a two-component scheme);

10. Water - the rest.

When growing the strain Rhodococcus rhodochrous M33 under conditions of periodic cultivation in flasks, glucose was added to the MS and MS-1 media as a carbon and energy source, maintaining its concentration in the range 12.0–120.0 g / L. Glucose is introduced into the culture medium in one or two parts. When applied in two parts, glucose is additionally (fractionally) added after 30-40 hours from the start so that the total amount of glucose added does not exceed 120.0 g / l.

When cultured in laboratory or industrial fermenters according to a two-component scheme, sodium acetate and acetic acid can be used as the second carbon source. In this case, at the first stage of growth, the process proceeds using glucose. Then, when glucose ends, sodium acetate is added to the apparatus to a final concentration of 2.0-6.0 g / l, followed by maintaining the pH of the medium at a given level (usually 7.2-8.0) with a 50% aqueous solution acetic acid.

Measurement of nitrile hydratase activity was carried out as follows.

ml of a solution of acrylonitrile with a concentration of 2% in 10 mm phosphate buffer (pH 7.5) was mixed with 1 ml of a suspension of Rhodococcus rhodochrous M33 cells obtained by preliminary centrifugation

- 5 032896 lowering the culture fluid, washing and then resuspending the cells in 10 mM phosphate buffer (pH 7.5). The suspension contained from 0.08 to 0.16 mg of cells (dry weight). The reaction proceeded for 10 minutes at 20 ° C with constant stirring, after which it was stopped by the addition of 50 μl of concentrated HCl. The concentration of acrylamide formed was determined by gas chromatography or photometry.

Nitrile hydratase activity is given in the following units:

specific activity - in micromoles of acrylamide per 1 minute per 1 milligram of dry biomass [U / mg];

total activity - in micromoles of acrylamide per 1 minute per milliliter of culture fluid [U / ml].

Inoculum preparation was carried out according to the following technology.

Rhodococcus rhodochrous M33 strain cells were grown on beveled meat-peptone agar at 30 ° C for 24 hours and suspended in 3 ml of physiological saline. The resulting suspension in a volume of 1 ml was inoculated with a 250 ml Erlenmeyer flask containing 50 ml of MS medium of the following composition (g / l): Na ₂ HPO ₄ -12H ₂ O - 7.9; KH ₂ PO ₄ - 1.8; FeSO ₄ -7H ₂ O - 0.1; EDTA Na - 0.2; CoCl ₂ -6H ₂ O - 0.01; MgSO ₄ -7H ₂ O - 1.0; urea - 4.0; glucose - 12.0. The cultivation was carried out with stirring on a circular shaker and 30 ° C for 48 hours. The resulting culture liquid was used to inoculate flasks and fermenters in all subsequent experiments.

It should be understood that these and all examples cited in the application materials are not limiting and are provided merely to illustrate the present invention.

Example 1. Cultivation of a bacterial strain of Rhodococcus rhodochrous M33 at various concentrations of a carbon source under laboratory conditions.

250 ml Erlenmeyer flasks containing 50 ml of MS medium of the following composition (g / l): Na ₂ HPO ₄ -12H ₂ O - 7.9; KH ₂ PO ₄ - 1.8; FeSO ₄ -7H ₂ O - 0.1; EDTA Na - 0.2; CoCl ₂ -6H ₂ O - 0.06; MgSO ₄ -7H ₂ O 1.0; urea - 16.0; glucose - 20.0-120.0, inoculated with 0.5 ml of inoculum. The cultivation was carried out with stirring on a circular shaker at 180 rpm and 30 ° C. The total yield of biomass and nitrile hydratase activity of the cells were determined. The data obtained are given in table. 1.

As can be seen from the table. 1, with an increase in glucose concentration from 20 to 120 g / l in the medium, an increase in the yield of biomass and total nitrile hydratase activity occurs.

Table 1

Glucose concentration (g / l) The yield of dry biomass (g / l) Growth time (h) Nitrile hydratase activity Specific Activity (U / ml) Total activity (U / ml) 20 7.6 72 241.2 1833.1 thirty 11.6 72 249.2 2890.1 40 15.8 72 258.9 4090.6 fifty 20 72 277.4 5548 60 24 96 312.5 7500 70 28.6 96 284.9 8148.1

............

Example 2. Comparative cultivation of the bacterial strain Rhodococcus rhodochrous M33 under laboratory conditions.

A 250 ml Erlenmeyer flask containing 50 ml of MS medium of the following composition (g / l): Na2HPO4-12H2O - 7.9; KH2PO4 - 1.8; FeSO4-7 ^ O - 0.1; EDTA Na - 0.2; CoC ^^ O - 0.06; MgSO4-7 ^ O 1.0; urea - 16.0; glucose - 40.0, and a 250 ml Erlenmeyer flask containing 50 ml of MS1 medium of the following composition (g / l): Na ₂ HPO ₄ -12H ₂ O - 7.9; KH ₂ PO ₄ - 1.8; aspartate Fe ²⁺ - 0.11; EDTA Na - 0.2; aspartate Co ²⁺ - 0.084; aspartate Mg ²⁺ 1.17; urea - 16.0; glucose - 40.0, inoculated with 0.5 ml of inoculum. The cultivation was carried out with stirring on a circular shaker at 180 rpm and 30 ° C for 96 hours. The total yield of biomass and nitrile hydratase activity of the cells were determined. The data obtained are given in table. 2.

As can be seen from table 2, the replacement of inorganic salts of iron, cobalt and magnesium with the corresponding aspartates not only increases the total nitrile hydratase activity by 12%, but also prevents the decrease in specific nitrile hydratase activity in the stationary phase.

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table 2

Growth time (h) MS environment MS-1 environment The yield of dry biomass (g / l) Nitrile hydratase activity The yield of dry biomass (g / l) Nitrile hydratase activity Specific Activity (U / ml) Total activity (U / ml) Specific Activity (U / ml) Total activity (U / ml) 24 1.8 88 158.4 1.8 86.6 155.9 48 12.6 122.7 1546 12.6 117.2 1476.7 72 15.8 258.9 4090.6 16 290.2 4643,2 96 14 179.4 2511.6 fifteen 234.3 3514.5

Example 3. The cultivation of the bacterial strain Rhodococcus rhodochrous M33 on a medium containing aspartic acid in laboratory conditions.

Into an Erlenmeyer flask with a volume of 250 ml containing 50 ml of MS medium of the following composition (g / l): Na ₂ HPO ₄ -12H ₂ O - 7.9; KH ₂ PO ₄ - 1.8; FeSO ₄ -7H ₂ O - 0.1; EDTA Na - 0.2; CoCl ₂ -6H ₂ O - 0.06; MgSO ₄ -7H ₂ O 1.0; urea - 16.0; glucose - 40.0, 2 g / l of aspartic acid were added. The flask was inoculated with 0.5 ml of inoculum. The cultivation was carried out with stirring on a circular shaker at 180 rpm and 30 ° C. After 72 hours of cultivation, 16.2 g / L dry cells with a nitrile hydratase activity of 290.3 U / mg were obtained. The total nitrile hydratase activity was 4702.9 U / ml.

Thus, the introduction of aspartic acid into the culture medium led to an increase in nitrile hydratase activity and yield of dry cells by 12.1 and 2.5%, respectively. Total nitrile hydratase activity increased by 15%.

Example 4. Cultivation of the bacterial strain Rhodococcus rhodochrous M33 on a medium containing aspartic acid in a laboratory fermenter.

A three-liter laboratory fermenter containing 1.5 l of MS medium of the following composition (g / l): Na ₂ HPO ₄ -12H ₂ O - 7.9; KH ₂ PO ₄ - 1.8; FeSO ₄ -7H ₂ O - 0.1; EDTA Na - 0.2; CoCl ₂ -6H ₂ O - 0.06; MgSO ₄ -7H ₂ O 1.0; urea - 16.0; glucose - 20.0; Aspartic acid 2.0, inoculated with 50 ml of inoculum. Cultivation was carried out at 30 ° C and aeration of 0.5 min ^-1 . The number of revolutions of the mixer was adjusted so that the concentration of dissolved oxygen was maintained at 50%. When glucose in the medium was over, 4 g / l sodium acetate was introduced into the apparatus and pH was maintained at 7.8 with a 50% aqueous solution of acetic acid. After 72 hours of cultivation, 22.3 g / L dry cells with a nitrile hydratase activity of 340.6 U / mg were obtained. The total nitrile hydratase activity was 7595.4 U / ml.

Example 5. Cultivation of a bacterial strain Rhodococcus rhodochrous M33 with fractional introduction of a carbon source in a laboratory fermenter.

A three-liter laboratory fermenter containing 1.5 l of MS medium of the following composition (g / l): Na ₂ HPO ₄ -12H ₂ O - 7.9; KH ₂ PO ₄ - 1.8; FeSO ₄ -7H ₂ O - 0.1; EDTA Na - 0.2; CoCl ₂ -6H ₂ O - 0.06; MgSO ₄ -7H ₂ O 1.0; urea - 16.0; glucose - 40.0; Aspartic acid 2.0, inoculated with 50 ml of inoculum. Cultivation was carried out at 30 ° C and aeration of 0.5 min ^-1 . The number of revolutions of the mixer was adjusted so that the concentration of dissolved oxygen in the medium was maintained at 50%. For 30 and 40 hours of growth, another 40 g / L of glucose in the form of a 40% solution was added to the apparatus. The total glucose concentration was 120 g / l. After 72 hours of cultivation, 45.1 g / L dry cells with a nitrile hydratase activity of 189.8 U / mg were obtained. The total nitrile hydratase activity was 8560 U / ml.

Example 6. Cultivation of the bacterial strain Rhodococcus rhodochrous M33 with fractional introduction of a carbon source in an industrial environment.

An industrial fermenter with a volume of 50 m ³ containing 35 m ³ of MS medium of the following composition (g / l): H ₃ PO ₄ (100%) - 3.45; KOH-0.7; NaOH - 2.0; FeSO ₄ -7H ₂ O - 0.1; EDTA Na - 0.2; CoCl ₂ -6H ₂ O - 0.06; MgSO ₄ -7H ₂ O - 1.0; urea - 16.0; glucose - 40.0; Aspartic acid 2.0, inoculable 1.0 m ³ inoculum. Fermentation was carried out at 28-30 ° C and aeration of 0.5 min ^-1 .

For 30 hours of growth, another 20.0 g / l glucose was introduced into the apparatus. After 64 hours of cultivation, 23 g / L dry cells with a specific nitrile hydratase activity of 296 U / mg were obtained. Total activity was 6808 U / ml.

Example 7. Cultivation of the bacterial strain Rhodococcus rhodochrous M33 using two carbon sources in an industrial environment.

An industrial fermenter with a volume of 50 m ³ containing 35 m ³ of MS medium of the following composition (g / l): H ₃ PO ₄ (100%) - 3.45; KOH - 0.7; NaOH - 2.0; FeSO ₄ -7H ₂ O -0.1; EDTA Na - 0.2; CoCl ₂ -6H ₂ O - 0.06; MgSO ₄ -7H ₂ O - 1.0; urea - 16.0; glucose - 40.0; Aspartic acid 2.0, inoculable 1.0 m ³ inoculum. Fermentation was carried out at 28-30 ° C and aeration of 0.5 min ^-1 . For 26 hours of growth, they introduced another

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20.0 g / l glucose. When the glucose in the medium was over, 4 g / L sodium acetate was added to the apparatus and the pH was further maintained at 7.8 with a 50% aqueous solution of acetic acid. After 72 hours of cultivation, 24.0 g / L dry cells with nitrile hydratase activity of 304.2 U / mg were obtained. The total nitrile hydratase activity was 7300.8 U / ml.

The proposed method provides a more efficient industrial production of biomass of producer strains of Rhodococcus rhodochrous M33 with high total nitrile hydratase activity. This eliminates the need for the use of growth factors and expensive inductors.

Although the invention has been described with reference to the disclosed embodiments, it should be apparent to those skilled in the art that the specific experiments described in detail are for illustrative purposes only and should not be construed as in any way limiting the scope of the invention. It should be understood that various modifications are possible without departing from the gist of the present invention.

Claims (21)

CLAIM 1. The method of cultivation of Rhodococcus rhodochrous M33 - a producer of nitrile hydratase, characterized in that the microorganism is cultured in a medium including anions of aspartic acid, as well as metal ions - iron, magnesium and cobalt. 2. The method according to claim 1, characterized in that the sources of metal ions are metal aspartates and / or inorganic metal salts. 3. The method according to claim 2, characterized in that the source of iron, magnesium or cobalt ions are, respectively, iron (II) aspartate, iron (III) aspartate, magnesium (II) aspartate or cobalt (II) aspartate. 4. The method according to claim 2, characterized in that the inorganic metal salt is sulfate, chloride and / or their hydrates. 5. The method according to claim 4, characterized in that the inorganic salt is FeSO ₄ , MgSO ₄ , CoCl ₂ and / or their hydrates. 6. The method according to claim 1, characterized in that the source of anions of aspartic acid is aspartic acid, iron aspartate, magnesium aspartate and / or cobalt aspartate. 7. The method according to claim 1, characterized in that the source of anions of aspartic acid is aspartic acid, and the source of at least one of the metal ions is its inorganic salt. 8. The method according to claim 1, characterized in that the total concentration of anions of aspartic acid in the medium is 0.076-3.0 g / L. 9. The method according to claim 1, characterized in that in the process of cultivation, after 30-40 hours from the start of cultivation, glucose is additionally added to the medium so that the total amount of glucose in the medium does not exceed 120.0 g / l. 10. The method according to claim 1, characterized in that sodium acetate is additionally added to the culture medium so that the total amount of sodium acetate added does not exceed 6.0 g / l. 11. The method according to claim 10, characterized in that the pH of the medium is maintained at a predetermined level by a solution of acetic acid. 12. The culture medium for Rhodococcus rhodochrous M33 is a nitrile hydratase producer, characterized in that it includes a source of aspartic acid anions, as well as sources of metal ions - iron, magnesium and cobalt. 13. The cultivation medium of claim 12, wherein the metal ion sources are metal aspartates and / or inorganic metal salts. 14. The culture medium of claim 13, wherein the source of iron, magnesium or cobalt ions are, respectively, iron (II) aspartate, iron (III) aspartate, magnesium (II) aspartate or cobalt (II) aspartate. 15. The culture medium according to item 13, in which the inorganic salt is a sulfate, chloride and / or their hydrates. 16. The culture medium according to clause 15, in which the inorganic salt is FeSO ₄ , MgSO4, CoCl2 and / or their hydrates. 17. The culture medium of claim 12, wherein the source of aspartic acid anions is aspartic acid, iron aspartate, magnesium aspartate and / or cobalt aspartate. 18. The cultivation medium according to claim 12, wherein the source of aspartic acid anions is aspartic acid and the source of at least one of the metal ions is its inorganic salt. 19. The culture medium according to item 12, in which the total concentration of anions of aspartic acid is 0.076-3.0 g / L. 20. The culture medium according to item 12, containing, g / l: Na ₂ HPO ₄ -12H ₂ O - 2.2-13.4; - 8 032896 KH ₂ PO ₄ - 0.5-3.1; FeSO ₄ -7H ₂ O - 0.01-0.3; EDTA Na - 0.02-0.6; CoCl ₂ -6H ₂ O - 0.01-0.08; MgSO ₄ -7H ₂ O - 0.05-2.5; urea - 4.0-20.0; glucose - 12.0-120.0; aspartic acid - 0.076-3.0; water is the rest. 21. The culture medium according to item 12, containing, g / l: Na ₂ HPO ₄ -12H ₂ O - 2.2-13.4; KH ₂ PO ₄ - 0.5-3.1; iron (II) aspartate - 0.011-0.33; EDTA Na - 0.02-0.6; cobalt (II) aspartate - 0.014-0.11; magnesium aspartate (II) - 0.06-2.9; urea - 4.0-20.0; glucose - 12.0-120.0; water is the rest. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2